Increased production of secreted proteins by recombinant eukaryotic cells

ABSTRACT

Described herein are methods for increasing the amount of protein secreted by a cell. In one case, a cell is provided which contains a heterologous nucleic acid encoding a protein having unfolded protein response modulating activity and a heterologous nucleic acid encoding a protein of interest to be secreted. In one case, the protein having unfolded protein response modulating activity is selected from the proteins selected from the group consisting of HAC1, PTC2 and IRE1. The protein of interest can be any secreted protein such as a therapeutic or an industrial enzyme. For example the protein can be selected from the group consisting of lipase, cellulase, endo-glucosidase H, protease, carbohydrase, reductase, oxidase, isomerase, transferase, kinase, phosphatase, alpha-amylase, glucoamylase, lignocellulose hemicellulase, pectinase and ligninase.

RELATED APPLICATIONS

[0001] This application is a Continuation-In-Part of U.S. application Ser. No. 09/534,692, filed Mar. 24, 2000, and is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention relates to cells which have been genetically manipulated to have an elevated unfolded protein response (UPR) resulting in an increased capacity to produce secreted proteins.

BACKGROUND OF THE INVENTION

[0003] The secretory pathway of eukaryotic organisms is of interest since cells can be engineered to secrete a particular protein of interest. The secretory pathway starts by translocation of the protein into the lumen of the endoplasmic reticulum (ER). In the ER the proteins fold into their final three-dimensional conformation and the core part of the N-glycans are attached to them. A quality control mechanism involving the proteins calnexin and calreticulin also resides in the ER, letting only completely folded proteins continue on the secretory pathway to the next compartment (Hammond and Helenius, 1995, Curr. Opinion Cell Biol. 7:523-529). Secretory proteins that do not fold properly are transported back to the cytoplasm by the translocation machinery and are degraded by the proteasome system (Wiertz et al., 1996, Nature 384:432-438).

[0004] The folding and glycosylation of the secretory proteins in the ER is assisted by numerous ER-resident proteins. The chaperones like Bip (GRP78), GRP94 or yeast Lhs1p help the secretory protein to fold by binding to exposed hydrophobic regions in the unfolded states and preventing unfavourable interactions (Blond-Elguindi et al., 1993, Cell 75:717-728). The chaperones are also important for the translocation of the proteins through the ER membrane. The foldase proteins like protein disulphide isomerase and its homologs and prolyl-peptidyl cis-trans isomerase assist in formation of disulphide bridges and formation of the right conformation of the peptide chain adjacent to proline residues, respectively. A machinery including many protein components also resides in the ER for the addition of the N-linked core glycans to the secretory protein and for the initial trimming steps of the glycans.

[0005] The levels of the chaperone and foldase proteins found in the ER are regulated at the transcriptional level. For each gene there is a basic level of transcription that can be increased in response to various stimuli. A large amount of secretory protein in the ER (secretory load) can induce the mammalian GRP78 gene, and this induction is mediated through the NF-κB transcription factor (Pahl and Baeuerle,1995, EMBO J. 14:2580-2588). Furthermore, the ER chaperone and foldase genes are upregulated when the amount of unfolded protein increases in the ER. This induction has been named unfolded protein response (UPR) and it has been described in mammalian cells, yeast and filamentous fungi (McMillan et al., 1994, Curr. Opinion in Biotechnol. 5:540-545). The induction can be caused by treatment of cells with reducing agents like DTT, by inhibitors of core glycosylation like tunikamycin or by Ca-ionophores that deplete the ER calcium stores. The promoters of mammalian and yeast genes regulated by UPR have a conserved sequence region called UPR element, where the transcription factor responsible for the induction binds.

[0006] When the unfolded protein response pathway is active, a signal is tranduced from the ER lumen to the transcription machinery in the nucleus. A protein implicated in the UPR induction is the IRE1 protein of yeast (Cox et al., 1993, Cell 73:1197-1206, Mori et al., 1993, Cell 74:143-156). It is large protein having a transmembrane segment anchoring the protein to the ER membrane. A segment of the IRE1 protein has homology to protein kinases and the C-terminal tail has some homology to RNAses. It is believed that the IRE1 protein may be the first component of the UPR signal transduction pathway, sensing the ER lumen for the presence of unfolded proteins and transmitting the signal eventually to a transcription factor inducing the ER-protein genes. It has been reported that the IRE1 protein oligomerizes and gets phosphorylated when the UPR is activated (Shamu and Walter, 1996, EMBO J. 15:3028-3039). Over-expression of the IRE1 gene in yeast leads to constitutive induction of the UPR (Id.). Phosphorylation of the IRE1 protein occurs at specific serine or threonine residues in the protein.

[0007] Another protein reportedly implicated in the regulation of the UPR pathway is PTC2, a yeast protein phosphatase encoded by the PTC2 gene (Welihinda et al., 1998, Mol. Cell. Biol. 18, 1967-1977). The IRE1 protein is phosphorylated when the UPR pathway is turned on (Shamu and Walter, 1996, EMBO J. 15:3928-3039), and PTC2 dephosphorylates the IRE1 protein and regulates the UPR.

[0008] It has further been reported that the yeast transcription factor mediating the UPR induction of the chaperone and foldase genes is the HAC1 protein (Cox and Walter, 1996, Cell 87:391-404, Sidrauski et al., 1996, Cell 87:405-413). It belongs to the bZIP family of transcription factors, having a basic DNA-binding region and a leucine zipper dimerisation domain. The binding of the HAC1 protein to the UPR element of ER-protein gene promoters has been demonstrated (Mori et al., 1998, J. Biol. Chem. 273: 9912-9920). The action of the HAC1 protein is regulated by its amount in the cells; none of the protein can be found in uninduced cells and upon UPR induction it appears rapidly. The HAC1 protein amount is dependent of the splicing of the respective mRNA. In uninduced conditions the intron present in the HAC1 gene close to the translation termination codon is not spliced off, and this intron prevents the formation of HAC1 protein by preventing the translation of the mRNA (Chapman and Walter, 1997, Curr. Biol. 7,850-859, Kawahara et al., 1997, Mol. Biol. Cell 8, 1845-1862). When UPR is induced, the intron is spliced and the mRNA is translated to form HAC1 protein that activates the promoters of its target genes. The HAC1 intron is spliced by an mechanism not currently described for any other system, involving the RNAse activity of the IRE1 protein and a tRNA ligase (Sidrauski and Walter, 1997, Cell 90, 1031-1039, Gonzales et al., 1999, EMBO J. 18, 3119-3132, Sidrauski et al., 1996, Cell 87, 405-413). The unfolded protein response can be induced constitutively in yeast by transformation with a UPR inducing version of the HAC1 gene. (Cox and Walter, supra.)

[0009] Thus, as indicated above, there are a number of reports regarding the secretory pathway. Additionally, there are reports on how to increase secretion so as to provide greater yields of heterologous proteins. Greater yields of protein are generally of interest to industry to provide more of a particular protein and to facilitate purification.

[0010] For example, in one report random mutagenesis of the host organism has been performed followed by screening for increased yield of a secreted protein. In another report, there has been fusion of a heterologous protein to an efficiently secreted endogenous protein in order to increase the yield of secretion of the heterologous protein. Both of these methods have been of limited success and other methods to improve protein secretion are desirable.

[0011] In other studies, there has reportedly been increased yields of secreted heterologous proteins in yeast by either over-expression or deletion of the yeast ER foldase or chaperone genes on an individual or pairwise basis. For example, over-expression of either the protein disulphide isomerase (PDI) or the KAR2 (homologous to the gene for the mammalian ER chaperone BiP) genes in yeast has been shown to increase the extracellular accumulation of certain secreted heterologous proteins (Robinson et al., 1996, Bio/Technology, 12:381-384; Harmsen, et al., 1996, Appl. Microbiol. Biotechnol., 46:365-370). In contrast, deletion of the CNE1 gene, encoding an ER chaperone homologous to mammalian calnexin, reportedly can lead to increased secretion of a heterologous protein (Parlati et al., 1995, J. Biol. Chem. 270:244-253, Harmsen, supra.). The effect of over-expression or deletion of individual or pairs of ER chaperones or foldases has also been reported on in filamentous fungi, however, increased secretion was not obtained. (Punt, et al., 1998, Appl. Microbiol. Biotech, 50:447-454; Wang, et al., 2000, Current Genetics, 37:57-64).

[0012] Therefore, it is desirable to provide new methods to increase production of secreted proteins in eukaryotic cells which are simple and consistent. It is also desirable to provide compositions such as novel genes to be used in methods for the increased production of secreted proteins. It is further desirable to provide. eukaryotic cells according to the invention which are transformed with heterologous genes so as to have an increased capacity to produce secreted proteins.

SUMMARY OF THE INVENTION

[0013] Provided herein are methods for increasing the secretion of a heterologous protein in a cell comprising inducing an elevated unfolded protein response (UPR). The increase in protein secretion is compared to a level of protein secreted by the cell when the UPR is not elevated by the methods described herein. In one aspect, the method includes inducing the elevated UPR by increasing the presence of the HAC1 protein in the cell. In one aspect of the invention, the presence of the HAC1 protein can be increased by a number of methods. For example, the HAC1 gene can be overexpressed compared to its native state. Overexpression can be achieved by a variety of ways including the use of preferred vectors and promoters as further described herein. In one embodiment, the HAC1 protein is increased in a cell by transformation of said cell by a nucleic acid comprising a UPR inducing form of a HAC1 recombinant nucleic acid.

[0014] The HAC1 nucleic acid encoding a HAC1 protein can be from a variety of sources. It is understood that in one embodiment, HAC1 is used interchangeably with hac1, hacA, etc., and one embodiment is meant to encompass HAC1 homologs. Additionally, the skilled artisan can ascertain by the context whether the HAC1 is a nucleic acid, protein or either. In one embodiment, a HAC1 nucleic acid is isolated from yeast. In another embodiment, a HAC1 nucleic acid is isolated from filamentous fungi such as Trichoderma or Aspergillus.

[0015] In another aspect of the invention, the elevated UPR is induced by modulating the levels of IRE1 protein or PTC2 protein in said cell. Nucleic acids encoding IRE1 or PTC2 can be isolated from yeast or filamentous fungi such as Trichoderma or Aspergillus. In a preferred embodiment the nucleic acid encoding IRE1 or PTC2 is isolated from T. reesei, A. nidulans or A. niger.

[0016] The cell from which the protein is secreted can be any cell having an UPR. Cells having an UPR include all eukaryotes including but not limited to mammalian cells, insect cells, yeast and filamentous fungi.

[0017] Also provided herein is an isolated nucleic acid encoding a HAC1 protein, wherein said HAC1 has unfolded protein response inducing activity and has less than 50% similarity to yeast HAC1 protein. In another embodiment, an isolated nucleic acid encoding a HAC1 protein is provided, wherein said HAC1 protein has unfolded protein response inducing activity and wherein said HAC1 comprises a DNA binding region that has greater than 70% similarity to the DNA binding region of filamentous fungi HAC1. Embodiments of a DNA binding region are shown at amino acids 84-147 of the T. reesei protein shown in FIG. 10, at amino acids 53-116 of the A. nidulans protein shown in FIG. 10, and at amino acids 45-109 of the A. niger protein shown in FIG. 28. In one embodiment, the HAC1 protein encoded by the HAC1 nucleic acid provided herein has an amino acid sequence having greater than 70% similarity to the sequence of FIG. 7, FIG. 8 or FIG. 28. The proteins encoded by such nucleic acids are also provided herein.

[0018] In one embodiment, the nucleic acid provided herein encodes an amino acid sequence as set forth in FIG. 7, FIG. 8 or FIG. 28. In yet another embodiment, the nucleic acid provided herein has a nucleic acid sequence as set forth in FIG. 7, FIG. 8 or FIG. 28. The proteins encoded by such nucleic acids are also provided herein.

[0019] Further provided herein is an isolated nucleic acid encoding a PTC2 protein wherein said PTC2 protein modulates unfolded protein response and wherein said PTC2 protein has at least 70% similarity to the amino acid sequence of FIG. 24 or FIG. 25. In preferred embodiments the PTC2 protein has preferably at least 80%, more preferably at least 90% or more preferably at least 95% similarity to said amino acid sequences. In one aspect, the PTC2 protein has an amino acid sequence as set forth in FIG. 24 or FIG. 25. In another aspect, the PTC2 nucleic acid has a nucleic acid sequence as set forth in FIG. 24 or FIG. 25. The proteins encoded by such nucleic acids are also provided herein. It is understood that as used herein, PTC2 can be used interchangeably with ptc2 and ptcB, and that in one embodiment, PTC2 encompasses homologs. Moreover, the context in which the term is used will determine whether PTC2 is a nucleic acid, a protein or either.

[0020] Also provided herein is a nucleic acid encoding an IRE1 protein having unfolded protein response modulating activity and having at least 60% similarity to an amino acid sequence as shown in FIG. 26 or FIG. 27. In preferred embodiments the IRE1 protein has at least 70%, preferably at least 80%, more preferably at least 90% or even more preferably at least 95% similarity to said amino acid sequences. In one aspect, IRE1 has an amino acid or nucleic acid sequence as shown in FIG. 26 or FIG. 27. It is understood that as used herein, IRE1, Ire1 and IreA can be used interchangeably, and that in one embodiment, IRE1 includes homologs. Moreover, the context in which the term is used will determine whether IRE1 is a nucleic acid, a protein or either.

[0021] The nucleic acids provided herein may be obtained from a variety of sources including but not limited to filamentous fungi such as Trichoderma and Aspergillus. In a preferred embodiment the nucleic acids are obtained from T. reesei, A. nidulans or A. niger.

[0022] Also provided herein is a cell containing a heterologous nucleic acid encoding a protein having unfolded protein response modulating activity and a heterologous nucleic acid encoding a protein of interest to be secreted. In one aspect, said protein having unfolded protein response modulating activity is selected from the group consisting of HAC1, PTC2 and IRE1. In another embodiment, said protein of interest is selected from the group consisting of lipase, cellulase, endo-glucosidase H, protease, carbohydrase, reductase, oxidase, isomerase, transferase, kinase, phosphatase, alpha-amylase, glucoamylase, lignocellulose hemicellulase, pectinase and ligninase.

[0023] Further aspects of the invention will be understood by the skilled artisan as further described below.

BRIEF DESCRIPTION OF THE FIGURES

[0024]FIG. 1 depicts a map of the plasmid pMS109, an embodiment of a plasmid constructed for the expression of the truncated yeast HAC1 gene.

[0025]FIG. 2 depicts a graph showing activity of a-amylase produced from yeast containing pMS109 (squares) or an empty control vector pKK1 (diamonds) in the vertical bar, over time, horizontal bar, and further showing the activity is greater wherein pMS109 is present.

[0026]FIG. 3 depicts a bar graph showing activity of invertase produced from yeast containing pMS109 (black bars) or an empty control vector pKK1 (shaded bars) in the vertical bar, over time, horizontal bar, and further showing the activity is greater wherein pMS109 is present.

[0027]FIG. 4 depicts a graph showing activity of a-amylase produced from yeast wherein HAC1 has been disrupted (diamonds) or from its parental control strain (squares) in the vertical bar, overtime, horizontal bar, and further showing that the activity is greater wherein HAC1 has not been disrupted.

[0028]FIG. 5 depicts a graph showing activity of Trichoderma reesei (T. Reesei) endoglucanase EGI produced from yeast wherein HAC1 has been disrupted (diamonds) or from its parental control strain (squares) in the vertical bar, over time, horizontal bar, and further showing that the activity is greater wherein HAC1 has not been disrupted.

[0029]FIG. 6 depicts a map of the plasmid pMS119, where the full-length T. reesei HAC1 cDNA without the. 20 bp intron is in the pBluescript SK⁻ vector.

[0030]FIG. 7 depicts an embodiment of a nucleotide (SEQ ID No. 1) and deduced amino acid sequence (SEQ ID No. 2) of T. reesei HAC1. The introns are shown in lower case letters.

[0031]FIG. 8 depicts an embodiment of a nucleotide (SEQ ID No. 3) and deduced amino acid sequence (SEQ ID No. 4) of Aspergillus nidulans (A. nidulans) hacA. The introns are shown in lower case letters.

[0032]FIG. 9 depicts the hairpin loop structures forming at the 5′ end of the 20 bp introns in the T. reesei HAC1 and A. nidulans hacA mRNAs and at the 3′ end of the intron of the S. cerevisiae HAC1 mRNA. The conserved nucleotides in the loop region are shown in bold. The cleavage site of the yeast intron and the three possible cleavage sites of the T. reesei HAC1 intron are shown by arrows. Alignment of the 20 bp intron areas of the T. reesei HAC1 and A. nidulans hacA is shown below. The intron is in lower case.

[0033]FIG. 10 depicts an amino acid sequence alignment of the T. reesei HAC1, A. niduans hacA and S. cerevisiae HAC1 proteins. Identical amino acids are shown by asterisks and similar ones by dots. Yeast HAC1 is homologous to the other sequences at the DNA binding domain area. The DNA binding domain is approximately at amino acids 84-147 for T. reesei (SEQ ID No. 5), and approximately at amino acids 53-116 for A. nidulans (SEQ ID No. 6).

[0034]FIG. 11 depicts Northern hybridization of RNA samples derived from T. reesei mycelia treated with DTT (+DTT) and untreated control mycelia (−DTT). The timepoints (in minutes) after DTT addition are shown. The probes used for hybridization are shown on the left.

[0035]FIG. 12 depicts Northern hybridization of RNA samples derived from A. nidulans mycelia treated with DTT (+DTT) and untreated control mycelia (−DTT). The timepoints after DTT addition are shown. The probes are shown on the left.

[0036]FIG. 13 depicts a map of the plasmid pMS131, where the full-length T. reesei HAC1 cDNA without the 20 bp intron is under the yeast PGK1 promoter in the vector pAJ401.

[0037]FIG. 14 depicts a map of the plasmid pMS132, where the T. reesei HAC1 cDNA without the 5′ flanking region and without the 20 bp intron is under the yeast PGK1 promoter in the vector pAJ401.

[0038]FIG. 15 depicts complementation of S. cerevisiae HAC1 and IRE1 disruptions (DHAC1 and DIRE1, respectively) with different forms of the T. reesei HAC1 cDNA. The growth of transformants on media with and without inositol is shown. pAJ401 is the expression vector without any insert. pMS131 has the full-length T. reesei HAC1 cDNA in pAJ401. pMS132 has the T. reesei HAC1 cDNA without its 5′ flanking region in pAJ401.

[0039]FIG. 16 depicts bandshift experiments, where the binding of the maIE-HAC1 fusion protein to the putative UPR element sequences found in T. reesei pdi1 and bip1 promoters was tested. The oligonucleotides used in the binding reactions are shown on the top. Lanes 1, 12 and 16, no protein; lanes 2, 4-7, 8-11, 13-15 and 17-19, maIE-HAC1 fusion protein; lane 3, maIE protein alone. The binding was competed with unlabelled oligonucleotides on lanes 5 (20×excess); lanes 6, 10, 14 and 18 (50×excess) and lanes 7, 11, 15, and 19 (200×excess). Alignment of the UPR element sequences that bind the HACI-maIE protein is shown below.

[0040]FIG. 17 depicts a graph which shows activity of α-amylase by yeast strains expressing the T. reesei HAC1 cDNA without the 5′ flanking region and the 20 bp intron (pMS132) (squares) and control strains with the expression vector alone (pAJ401) (diamonds) in the vertical bar over time, horizontal bar, and which further shows that activity is greater wherein pMS132 is present.

[0041]FIG. 18 depicts a bar graph which shows activity of invertase by yeast strains expressing the T. reesei HAC1 cDNA without the 5′ flanking region and the 20 bp intron (pMS132) and control strains with the expression vector alone (pAJ401) in the vertical bar, over time (horizontal bar) and which further shows that activity is greater is greater wherein pMS132 is present.

[0042]FIG. 19 depicts Northern hybridization of RNA samples from a yeast strain expressing the T. reesei HAC1 cDNA without the 5′ flanking region and the 20 bp intron (pMS132) and a control strain with the expression vector alone (pAJ401). The probes used for hybridization are shown. The signals were quantified with a phosphoimager and the KAR2 signal intensities were normalised with respect to the TDH1 signal intensities. The normalised KAR2 signals are shown on the bottom wherein it is shown that pMS132 has greater signal.

[0043]FIG. 20 depicts a map of the plasmid pMS136, where the T. reesei HAC1 cDNA without the 5′ flanking region and the 20 bp intron is under the A. nidulans gpdA promoter in the vector pAN52-NotI.

[0044]FIG. 21 depicts Northern hybridization of RNA samples derived from transformation of the plasmid pMS136 into a T. reesei strain producing CBHI-chymosin fusion protein. Samples from the parental strain (lanes 1, 5 and 9), two positive transformants (lanes 2, 3, 6, 7, 10 and 11) and a HAC1 mutant strain desginated number 31 generated in the transformation (lanes 4, 8 and 12) are shown. The growth times are shown on the top and the probes used for the hybridization on the left. Quantifications of the pdi1 and bip1 signals normalised with respect to gpd1 signals are shown on the bottom.

[0045]FIG. 22 depicts Northern hybridization of RNA samples derived from mycelia of the HAC1 mutant strain number 31 treated with DTT (+DTT) and untreated control mycelia (−DTT). The timepoints after DTT addition are shown on the top and the probes used for hybridization on the left. Quantifications of the pdi1 signals normalised with respect to gpd1 signals are shown on the bottom.

[0046]FIG. 23 is a graph depicting production of calf chymosin by the HAC1 mutant transformant number 31 (diamonds) and its parental strain (squares) during a shake flask culture. The chymosin (CHV) units per ml of culture are shown (vertical bar) over time (horizontal bar), and it is shown that the control has more units than the mutant.

[0047]FIG. 24 depicts an embodiment of a nucleotide (SEQ ID No. 7) and deduced amino acid sequence (SEQ ID No. 8) of the fragment isolated from the A. nidulans ptcB gene. The intron is shown in lower case.

[0048]FIG. 25 depicts an embodiment of a nucleotide (SEQ ID No. 9) and deduced amino acid sequence (SEQ ID No.10) of the T. reesei ptc2 cDNA.

[0049]FIG. 26 depicts an embodiment of a nucleotide (SEQ ID No. 11) and deduced amino acid sequence (SEQ ID No.12) of the fragment isolated from the A. nidulans ireA gene. The intron is shown in lower case.

[0050]FIGS. 27A-27C depict an embodiment of a nucleotide (SEQ ID No.13) and deduced amino acid sequence (SEQ ID No.14) of the T. reesei IRE1 gene. The intron is shown in lower case.

[0051]FIG. 28A-28C. The nucleotide (SEQ ID No.15) and deduced amino acid sequence (SEQ ID No. 16) of Aspergillus niger var. awamori hacA cDNA. The 20 bp unconventional intron (SEQ ID No. 17) is shown in lower case letters. The amino acid sequences of the upstream open reading frame (SEQ ID No. 18) and the HACA protein (SEQ ID No. 19) are shown below the nucleotide sequence.

[0052]FIG. 29. Map of the plasmid pMS152 where the Aspergillus niger var. awamori hacA without the 5′ flanking region and the 20 bp intron is under control of the Aspergillus niger var. awamori glaA promoter.

[0053]FIG. 30. The levels of chymosin activity measured in supernatants from duplicate cultures of strain ΔAP3pUCpyrGRG3#11 (ctrl) and transformants (#1, #2, #3 and #4) of this strain with pMS152.

[0054]FIG. 31. The levels of laccase activity measured in supernatants from duplicate cultures of strain ΔAP4:pGPTlaccase (ctrl) and transformants (#1, #2, #3, #4, #5, #6, #7 and #8) of this strain with pMS152.

DETAILED DESCRIPTION OF THE INVENTION

[0055] The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0056] Provided herein are methods and compositions for increasing the secretion of a protein in a cell comprising inducing an elevated unfolded protein response (UPR). The compositions provided herein include nucleic acids, proteins, and cells.

[0057] In one embodiment UPR refers to the unfolded protein response which occurs in response to an increase in unfolded protein in the ER. In a method provided herein, the UPR is elevated. In one embodiment, “elevated” UPR refers to an increase in the response compared to the response which would have been induced based on the amount of unfolded protein in the ER. In one embodiment, elevated refers to an increase with respect to the length of time the response occurs. In each embodiment, the elevated UPR results in an increased capacity for the cell to produce secreted proteins compared to another cell of the same type containing the same amount of unfolded protein in the ER. Preferably, the cell having an elevated UPR in accordance with the present invention produces more secreted protein in the same amount of time as a cell not having an elevated UPR.

[0058] In one aspect, the method includes inducing the elevated UPR by modulating the amount or presence of one or more UPR modulating proteins in said cell. In one embodiment, the UPR modulating protein is selected from the group consisting of HAC1, PTC2 or IRE1. UPR modulating proteins are further discussed below. It is understood that the modulating protein can be obtained by increasing the presence of a nucleic acid which encodes a modulating protein. The protein used in the methods herein have UPR modulating activity as further discussed below, and the nucleic acids encode a protein which has UPR modulating activity. Modulating means that an increase in the protein can lead to an increase or a decrease in the UPR. Thus, in one embodiment, the presence of a modulating protein is increased as further discussed below to reach an elevated UPR. In another embodiment, the modulating protein is decreased or eliminated to reach an elevated UPR. In a preferred embodiment, HAC1 and/or IRE1 are increased so as to reach an elevated UPR.

[0059] In one embodiment, inducing UPR means that the unfolded protein response as a whole is induced or maintained as it would be by unfolded protein in the ER. The unfolded protein response involves increased expression and regulation of multiple ER foldases and chaperones. Thus, in one embodiment, manipulation of ER foldases or chaperones on an individual gene basis would not be considered an induction of UPR. Thus, in a preferred embodiment, UPR modulating activity results in an elevated UPR wherein an elevated UPR results in upregulation of ER chaperones and foldases and increased secretion of proteins.

[0060] The nucleic acids encoding the UPR modulating proteins can be obtained from a variety of sources. Preferred organisms include but are not limited to Saccharomyces cerevisiae, Aspergillus spp. and Trichoderma spp. Also other suitable yeasts and other fungi, such as Schizosaccharomyces pombe, Kluyveromyces lactis, Pichia spp., Hansenula spp., Fusarium spp., Neurospora spp. and Penicillium spp. can be used. Homologous genes from other organisms can also be used. In one aspect, homologous genes refer to genes which are related, but not identical, in their DNA sequence and/or perform the same function are homologous with each other and are called each other's homologues.

[0061] HAC1, PTC2, or IRE1 amino acid and nucleic acid sequences have been described for yeast. For example, for HAC1, see GenBank accession number E15694; for PTC2, see GenBank accession number U72498; for IRE1, see GenBank accession number Z11701. Sequences of GenBank accession numbers are incorporated herein by reference. GenBank is known in the art, see, e.g., Benson, D A, et al., Nucleic Acids Research 26:1-7 (1998) and http://www.ncbi.nlm.nih.gov/. In one embodiment, HAC1, PTC2, or IRE1 are isolated from a species other than yeast, preferably a filamentous fungi, insect cell, mammalian cell or other eukaroyote. Sequences for HAC1 are provided in FIGS. 7, 8 and 28. Sequences for PTC2 are provided in FIGS. 24 and 25. Sequences for IRE1 are provided in FIGS. 26 and 27.

[0062] In one embodiment, the UPR modulating sequences are identified by hybridization to other nucleic acids. Additionally, sequence homology determinations can be made using algorithms.

[0063] Thus in one embodiment, the UPR modulating nucleic acid hybridizes to a complement of a nucleic acid encoding HAC1, PTC2 or IRE1. In one embodiment, the HAC1, PTC2 or IRE1 encoding sequence is selected from the sequences provided in the respective figures. In one embodiment the stringency conditions are moderate. In another embodiment, the conditions used are high stringency conditions.

[0064] “Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

[0065] “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

[0066] “Moderately stringent conditions” may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

[0067] Homologous (similar or identical) sequences can also be determined by using a “sequence comparison algorithm.” Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection.

[0068] An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.

[0069] The BLAST algorithm then performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a protein such as a protease if the smallest sum probability in a comparison of the test amino acid sequence to a protein such as a protease amino acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0070] In one embodiment, the HAC1 protein provided herein has less than 80% sequence similarity than the HAC1 yeast protein, see for example, GenBank accession number E15694, more preferably, less than 70%, more preferably, less than 60%, more preferably less than 50%, more preferably, less than 45% or 40% similarity. In another embodiment, identity is substituted for similarity.

[0071] In another embodiment, the HAC1 protein provided herein has at least 40% similarity to the amino acid sequence set forth in FIG. 7 or FIG. 8. More preferably, the similarity is at least 50%, more preferably, at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and more preferably at least 95% or 98%. In another embodiment, identity is substituted for similarity.

[0072] In another embodiment, the HAC1 protein provided herein comprises a DNA binding domain that has at least 70% similarity to the DNA binding domain set forth in FIG. 10. More preferably, the similarity is at least 70%, more preferably at least 80%, more preferably at least 90%, and more preferably at least 95% or 98%. In another embodiment, identity is substituted for similarity.

[0073] As used herein, DNA binding domain refers to the domain which binds to the conserved sequence called the UPR element in promoters of genes regulated by UPR. Embodiments of a DNA binding region are shown approximately at amino acids 84-147 of the T. reesei protein shown in FIG. 10, approximately at amino acids 53-116 of the A. nidulans protein shown in FIG. 10 and approximately amino acids 45-109 of the A. niger protein shown in FIG. 28. HAC1 homologs will have DNA binding domains which can be identified by activity or by alignment to the binding domains in FIG. 10.

[0074] In one embodiment, the PTC2 protein provided herein has less than 80% sequence similarity than the PTC2 yeast protein, see for example, GenBank accession number U472498, more preferably, less than 70%, more preferably, less than 60%, more preferably less than 50%, more preferably, less than 45% or 40% similarity. In another embodiment, identity is substituted for similarity.

[0075] In another embodiment, the PTC2 protein provided herein has at least 40% similarity to the amino acid sequence set forth in FIG. 24 or FIG. 25. More preferably, the similarity is at least 50%, more preferably, at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and more preferably at least 95% or 98%. In another embodiment, identity is substituted for similarity.

[0076] In one embodiment, the IRE1 protein provided herein has less than 80% sequence similarity than the IRE1 yeast protein, see for example, GenBank accession number Z11701, more preferably, less than 70%, more preferably, less than 60%, more preferably less than 50%, more preferably, less than 45% or 40% similarity. In another embodiment, identity is substituted for similarity.

[0077] In another embodiment, the IRE1 protein provided herein has at least40% similarity to the amino acid sequence set forth in FIG. 26 or FIG. 27. More preferably, the similarity is at least 50%, more preferably, at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, and more preferably at least 95% or 98%. In another embodiment, identity is substituted for similarity.

[0078] Additionally, further homologs of the UPR modulating sequences can be identified for example by using PCR primers based on the sequences provided herein. In yet another embodiment, naturally occurring allelic variants of the sequences provided herein may be used.

[0079] A protein has UPR modulating activity if it is able to regulate the induction of UPR. Regulate means causing an increase or decrease in the induction of the UPR. A UPR modulating protein can increase or decrease UPR induction whether or not there is a change in the amount unfolded protein in the ER. In a preferred embodiment, a UPR modulating protein has one or more of the following activities: HAC1 activity, PTC2 activity, IRE1 activity, or binds to HAC1.

[0080] Modulating the amount of or activity of the UPR modulating protein can occur by a variety of methods. For example, to increase the presence or activity of a protein in a cell, one can over-express the nucleic acid encoding the UPR modulating protein. Over-expression as used herein means that the protein encoded by the said gene is produced in increased amounts in the cell. In one embodiment, over-expression can be used interchangeably with constitutive expression or upregulation. This can be achieved by increasing the copy number of the gene by introducing extra copies of the gene into the cell on a plasmid or integrated into the genome. Over-expression can also be achieved by placing the gene under a promoter stronger than its own promoter. The amount of the protein in the cell can be varied by varying the copy number of the gene and/or the strength of the promoter used for the expression. Thus, manipulation of genes to cause induction of UPR may involve insertion into the host of multiple copies of a gene with its native promoter either on a replicating autosomal plasmid or by integration into the chromosomal DNA. It may involve fusion of the gene with a promoter region and/or transcriptional control sequences from other genes to further increase expression or to allow controlled, inducible expression. Agonists and enhancers may also be used.

[0081] In the case where it is desired to reduce the activity of a UPR modulating protein to result in elevated UPR, a number of methods may be used such as deletion of a gene or the use of antisense nucleic acids to reduce the expression of a gene. It may involve alteration of a gene to provide a mutant form of the protein or include the use of an inhibitor of a UPR modulating protein.

[0082] In one embodiment, UPR is elevated by using a UPR inducing form of a recombinant nucleic acid encoding a UPR-modulating protein. In one embodiment, a UPR-inducing form of a recombinant nucleic acid encoding a UPR-modulating protein is a nucleic acid which has been modified to give rise to a translatable mRNA. The translatable form mimics the modified mRNA which appears in the cell on induction of UPR and which can be translated to an active UPR-modulating protein.

[0083] In one embodiment, a UPR-inducing form of a recombinant nucleic acid includes coding sequence. Coding sequence as used herein includes the nucleic acid sequence which leads to the amino acid sequence of the protein in its active form. As used herein, a nucleic acid consisting essentially of a coding sequence explicitly excludes, lacks or omits at least internal sequence which does not get translated when the active protein is encoded. Internal sequence as used herein refers to sequence which is internal to the carboxyl terminus and the amino terminus. Examples of excluded internal sequence are shown in small letters in FIGS. 7, 8, 24, 26, 27 and 28. The sequence may be excluded by deletion or truncation by methods known in the art.

[0084] In one embodiment a nucleic acid comprises a sequence consisting essentially of coding sequence. In this embodiment, the nucleic acid may comprise vector sequence on either side of the coding sequence but the coding sequence excludes internal sequence which does not get translated in the encoded protein's active form.

[0085] In another embodiment, a UPR modulating protein is a variant UPR modulating protein which has been varied to have increased activity. Thus in one embodiment, the activity of a UPR modulating protein is increased to elevate UPR. In one embodiment, the activity of a UPR modulating protein is increased by maintaining the protein in its active state. For example, IRE1 is phosphorylated when the UPR pathway is turned on. Therefore, in one embodiment herein, maintaining IRE1 in its phosphorylated induces an elevated UPR.

[0086] In a preferred embodiment, IRE1 is mutated so as to constitutively have the activity of phosphorylated IRE1. In one embodiment, serine and/orthreonine residues are substituted with aspartic acid to form to form an IRE1 variant having constitutive UPR inducing activity. Other substitutions to mimic a protein in its phosphorylated state are known in the art. Preferably, the mutations are performed on the nucleic acid encoding the protein.

[0087] By the term “recombinant nucleic acid” herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by polymerases and endonucleases, in a form not normally found in nature. Generally, a nucleic acid refers to DNA, RNA or mRNA and includes a gene or gene fragment. Thus, an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.

[0088] Similarly, a “recombinant protein” is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above. Generally, the term protein and peptide can be used interchangeably herein. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure. For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample. A substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred. In one embodiment, the definition includes the production of a protein from other than its host cell, or produced by a recombinant nucleic acid. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.

[0089] A recombinant cell generally refers to a cell which has been manipulated to contain a recombinant nucleic acid or protein therein.

[0090] The protein of interest to be secreted can be any protein. Wherein the protein is not naturally secreted, the nucleic acid encoding the protein may be modified to have a signal sequence in accordance with techniques known in the art. The proteins which are secreted may be endogenous proteins which are expressed naturally, but in a greater amount in accordance with the present invention, or the proteins may be heterologous. In a preferred embodiment, the proteins are heterologous. Heterologous as used herein means the protein is produced by recombinant means. Therefore, the protein may be native to the cell, but is produced, for example, by transformation with a self replicating vector containing the nucleic acid encoding the protein of interest. Alternatively, recombinant could be wherein one or more extra copies of the nucleic acid are integrated into the genome by recombinant techniques.

[0091] In another embodiment, the protein of interest is selected from the group consisting of lipase, cellulase, endo-glucosidase H, protease, carbohydrase, reductase, oxidase, isomerase, transferase, kinase, phosphatase, alpha-amylase, glucoamylase, lignocellulose hemicellulase, pectinase and ligninase. In another embodiment, the protein of interest is a therapeutic selected from the group consisting of vaccines, cytokines, receptors, antibodies, hormones, and factors including growth factors.

[0092] The cell in which the proteins are secreted is any cell having an upregulated protein response. Preferably, the host to be transformed with the genes of the invention can be any eukaryotic cell suitable for foreign or endogenous protein production, e.g., any S. cerevisiae yeast strain, (e.g., DBY746, BMA64-1A, AH22, S150-2B, GYPY55-15bA, vtt-a-63015) any Trichoderma spp. such as T. Iongibrachiatum and the T. reesei strains derived from the natural isolate QM6a, such as RUTC-30, RL-P37, QM9416 and VIT-D-79125, any Kluyveromyces spp/. Sch. pombe, H. polymorpha, Pichia, Aspergillus, Neurospora, Yarrowia, Fusarium, Penicilium spp. or higher eukaryotic cells.

[0093] Examples of mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51).

[0094] In an alternative embodiment, a plant cell can be utilized. In another embodiment, a baculovirus infected insect cell is utilized. The selection of the appropriate host cell is deemed to be within the skill in the art.

[0095] Transfer of the genes into these cells can be achieved, for instance, by using the conventional methods of transformation described for these organisms. General aspects of mammalian cell host system transfections have been described in U.S. Pat. No. 4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, etc. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0096] The nucleic acid (e.g., cDNA, coding or genomic DNA) encoding the UPR modulating protein may be inserted into a replicable vector. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

[0097] For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces α-factor leaders, the latter described in U.S. Pat. No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the signal described in WO 90/13646 published 15 Nov. 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.

[0098] Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0099] Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

[0100] Transcription from vectors in mammalian host cells can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

[0101] Transcription of a DNA encoding the protein in eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5′ or 3′ to the coding sequence, but is preferably located at a site 5′ from the promoter.

[0102] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the protein. Still other methods, vectors, and host cells are described in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

[0103] In one embodiment, the gene is cloned into a suitable expression vector, such as pKK1 or similar vectors comprising the appropriate regulatory regions depending on the selected host. For example, these regulatory regions can be obtained from yeast genes such as the ADH1, GAL1-GAL10, PGK1, CUP1, GAP, CYC1, PHO5, or asparagine synthetase gene, for instance. Alternatively, also the regulatory regions of, for example, HAC1 can be used to express the gene in S. cerevisiae. The plasmid carrying the gene is capable of replicating autonomously when transformed into the recipient yeast strain and is maintained stably in a single copy due to the presence of a yeast centromeric sequence. Alternatively, a multicopy replicating plasmid could be used or integration of the plasmid into the yeast genomic DNA could be provided for using methods known in the art.

[0104] In one embodiment herein, to express HAC1 cDNA, preferably truncated in Trichoderma the coding region of the inducing form of the Trichoderma HAC1 gene is coupled for instance between the A. nidulans gpdA promoter and terminator and the expression cassette is transformed into a Trichoderma strain producing for instance bovine chymosin or another foreign protein. In the truncated form, the unconventional introns are removed, as well as any remaining terminal end adjacent to said intron. An uncoventional intron is one which is present in the mRNA in the cell which is not undergoing UPR, but which is removed from the mRNA upon induction of the UPR. UPR would be thus induced constitutively. A higher level of expression which was inducible according to the carbon source used for growth of the fungus could be achieved by fusion of the inducing form of HAC1 with the promoter of the T. reesei cbh1 gene.

[0105] For filamentous fungi the HAC1 gene is preferably integrated into the genome using methods known in the art. Suitable promoters in addition to the gpdA or cbh1 promoters or promoter of the HAC1 gene itself are for instance the other cellulase promoters, cbh2, egl1, egl2, or tef1, pgk, pki, the glucoamylase, alpha-amylase or the alcohol dehydrogenase promoter. In filamentous fungi transformation usually results in strains with varying copies of expression vector integrated into the genome (Penttilä et al., 1987) and from these the strain with optimal level of truncated HAC1 expression for growth and enhanced secretion can be screened.

[0106] It is understood that the methods provided herein may further include cultivating said recombinant host cells under conditions permitting expression of said secreted protein. The proteins can be collected and purified as desired. In a preferred embodiment, hydrolytic enzymes are secreted. In another embodiment, the secreted proteins are used in improved alcohol production or in processes where efficient hydrolysis of raw material is desired.

[0107] The following preparations and examples are given to enable those skilled in the art to more clearly understand and practice the present invention. They should not be considered as limiting the scope and/or spirit of the invention, but merely as being illustrative and representative thereof.

EXAMPLE 1 Effect of Expression of Truncated HAC1 in Yeast

[0108] In order to cause constitutive induction of the unfolded protein response in Saccharomyces cerevisiae, a truncated version of the yeast HAC1 gene was expressed from a centromeric plasmid. The truncated version does not include the intron of HAC1 that in normal conditions prevents the translation of the mRNA. Thus the mRNA expressed from the plasmid is translated to HAC1 protein constitutively and causes a constitutive induction of the unfolded protein response. The appropriate HAC1 gene fragment was first amplified from yeast chromosomal DNA by PCR. This fragment starts 24 bp before the translation start codon of the HAC1 gene and ends with a translation stop codon inserted after the proline codon at amino acid position 220 of the deduced protein. The oligonucleotide primers used were: 5′ ATC GCA GGA TTC CCA CCT ACG ACA ACA ACC GCC ACT 3′ (forward primer) (SEQ ID No. 20) and 5′ TAC AGC GGA TCC CTA TGG ATT ACG CCA ATT GTC AAG3′ (reverse primer) (SEQ ID No. 21). BamHI restriction sites were included into both of the primers to facilitate cloning. The PCR reaction was carried out with the Vent DNA polymerase (New England Biolabs) in conditions recommended by the manufacturer. The PCR program used started with heating to 94° C. for three minutes followed by 30 cycles with denaturation at 94° C. for 45 seconds, annealing at 55° C. for 45 seconds and synthesis at 72° C. for one minute. The PCR product fragment of 690 bp was run in a 0.8% agarose gel and purified from the gel by the Qiaquick gel extraction kit (Qiagen) according to manufacturer's protocol. The fragment was digested with BamHI and cloned into the BamHI site of the pZERO vector (Invitrogen) with methods known in the art. The HAC1 fragment was released from pZERO by BamHI digestion and cloned into the BgIII site of the vector pKK1 between the promoter and terminator of the yeast PGK1 gene with methods known in the art. pKK1 contains the LEU2 selectable marker gene and the centromere (CEN6) and ARS sequences for maintenance in yeast as a single-copy plasmid. The final expression plasmid was named pMS109 (FIG. 1).

[0109] The plasmid pMS109 and the control plasmid pKK1 were transformed into a yeast strain producing Bacillus amyloliquefaciens α-amylase. In this strain, the expression cassette with the a-amylase coding region inserted between the yeast ADH1 promoter and terminator had been integrated into the TRP1 locus of the yeast strain DBY746 (α, his3 1, leu2-3, ura3-52, trp1-289, Cyh^(r)). FourpMS109 transformants and four strains transformed with the vector pKK1 were selected for cultivations. The cultivation medium was synthetic complete yeast medium without leucine (S C-Leu, described by Sherman 1991, Meth. Enzymol. 194, 3-21), buffered to pH 6.0 with 2% succinic acid and supplemented with 2% glucose as the carbon source. The 50 ml yeast shake flask cultures were inoculated to the initial OD600 (optical density at the wavelength of 600 nm) of 0.2 and growth was carried out for five days at 30° C. and 250 RPM. Samples were taken daily for monitoring yeast growth and α-amylase production. α-amylase activity was measured with the Phadebas Amylase Test (Pharmacia) according to the instructions of the manufacturer. Yeast cell density was determined by measuring OD600 (optical density at the wavelength of 600 nm) of the culture. The α-amylase amounts produced by each of the pMS109 transformants were higher than the amounts produced by any of the pKK1 transformants. The average production level of pMS109 transformants was 70% higher in the end of the cultivation than the average of pKK1 clones (FIG. 2). The growth of the pMS109 strains was slightly retarded when compared with the control.

[0110] To analyse the effect of the constitutive UPR induction to yeast invertase production, four clones transformed with pMS109 and four clones transformed with the pKK1 vector, derived from the α-amylase producing strain described above, were cultivated in the SC-Leu medium buffered to pH 6.0 with 2% succinic acid and containing 2% sucrose as the carbon source. The 50 ml shake flask cultures were inoculated to the initial OD600 of 0.2 and grown subsequently for five days at 30° C. and 250 RPM. Yeast growth was followed by measuring the OD600 and samples were taken for invertase assays on days three, four and five. For each assay, cells were harvested by centrifugation from 1 ml of the culture. The cells were washed with 5 ml of 10 mM NaN₃ and resuspended in 0.2 M NaAc buffer, pH5.0 with 10 mM NaN₃. The invertase activity of the cells was measured by incubating them with 0.166 M sucrose in 0.2 M NaAc buffer, pH 5.0 for 6 minutes. The reaction was stopped by adding one volume of 0.5 M KPO₄, pH 7.0 and by separating the cells rapidly from the reaction mixture by filtration. The glucose formed into the reaction mixture was measured by the GOD-Perid kit (Boehringer Mannheim) according to the manufacturer's protocols. The invertase production of the pMS109 transformants was about 2 times higher than that of the pKK1 transformants in all the timepoints that were tested (FIG. 3).

EXAMPLE 2 Effect of Disruption of HAC1 in Yeast

[0111] The yeast HAC1 gene was disrupted by replacing it in the genome with a DNA fragment containing the G418 antibiotic resistance cassette flanked by 48 bp sequences from the 5′ and 3′ ends of the HAC1 open reading frame. The G418 resistance cassette consists of the E. coli kanamycin resistance gene cloned between the promoter and terminator of the Ashbya gossypii TEF gene encoding translation elongation factor 1. The DNA fragment used in the disruption of the yeast HAC1 was produced by PCR from the kanMX2 module (Wach et al.,1994, Yeast 10, 1793-1808) with the oligonucleotide primers 5′ CCA CCT ACG ACA ACA ACC GCC ACT ATG GAA ATG ACT GAT TTT GAA CTA CTT GCC TCG TCC CCG CCG GGT CAC 3′ (forward primer) (SEQ ID No. 22) and 5′ AAT TAT ACC CTC TTG CGA TTG TCT TCA TGA AGT GAT GAA GAA ATC ATT GAC ACT GGA TGG CGG CGT TAG TAT CGA 3′ (reverse primer) (SEQ ID No. 23). The PCR reaction was done with the Dynazyme DNA polymerase (Finnzymes) in conditions recommended by the manufacture. The PCR program started by denaturation at 94° C. for 3 minutes, followed by 30 cycles of denaturation at 94° C. for45 seconds, annealing at 52° C. for 30 seconds and elongation at 72° C. for 1 minute. A final elongation step of 5 minutes was performed at 72° C. The PCR product of about 1.5 kb was run in an 0.8% agarose gel and purified from the gel with the Qiaquick kit (Qiagen). The fragment was transformed into the yeast strain BMA64-1A (a, ura3-1, trp1-Δ, leu2-3, 112,his3-11, ade2-1, can1-100) with a method described (Gietz et al., 1992, Nucl. Acids Res. 20, 1425). The transformants were first grown over night on YPD plates (Sherman, 1991, Meth. Enzymol. 194, 3-21) and then replicated onto YPD plates with 200 μg/ml of the antibiotic G418. The transformants resistant to G418 were tested on plates with yeast mineral medium (Verduyn et al, 1992, Yeast 8, 501-517) with and without inositol. Chromosomal DNA was isolated from strains that were dependent on inositol, and Southern hybridization with the HAC1 protein-coding region was performed with methods known in the art. The result of the hybridization showed that the HAC1 gene had been disrupted in the strains dependent on inositol.

[0112] The effect of the HAC1 disruption on the production of two heterologous proteins, the Bacillus amyloliquefaciens α-amylase (Ruohonen et al., 1987, Gene 59, 161-170) and the Trichoderma reesei endoglucanase EGI (Penttilä et al., 1987, Yeast 3, 175-185), was tested. The α-amylase was expressed from a multicopy plasmid with the LEU2 marker gene, B485 (Ruohonen et al., 1991, J. Biotechnol. 39, 193-203, the plasmid is called YEpαa6 in this article), where the α-amylase gene has been cloned between the yeast ADH1 promoter and terminator. The EGI was expressed from the plasmid pMP311 (Penttilä et al., 1987, Yeast 3, 175-185), where the endoglucanase cDNA has been cloned between the yeast PGK1 promoter and terminator in a multicopy vector with the LEU2 marker gene. The B485 and pMP311 plasmids were transformed into the HAC1 disruptant and its parental strain with a described method (Gietz et al., 1992, Nucl. Acids Res. 20, 1425), and transformants were selected on SC-Leu plates (Sherman, 1991, Meth. Enymol. 194, 3-21). Three B485 transformants derived both from the HAC1 disruptant and its parental strain were grown in 50 ml shake flask cultures in SC-Leu buffered to pH 6.0 with 2% succinic acid and supplemented with 2% glucose. The cultures were inoculated to the initial OD600 of 0.2, and growth was continued for four days at 30° C. and 250 RPM. The α-amylase activity in the culture supernatants was assayed as described in Example 1. The HAC1 disruptant strain produced less than 10% of the α-amylase amount produced by the wild type control strain (FIG. 4). To test the effect on EGI production, three pMP311 transformants derived from the HAC1 disruptant and three transformants derived from the parental strain were grown in 50 ml of SC-Leu (Sherman, 1991, Meth. Enzymol. 194, 3-21) with 2% glucose in shaker flasks. The cultures were inoculated to the initial OD600 of 0.2, and grown at 30° C. and 250 RPM for four days. Endoglucanase activity of the cultures was measured with the substrate 4-methylumbelliferyl-β-D-lactoside (Sigma). Supernatant samples were incubated at 50° C. for 3 hours in a reaction mixture of 0.25 mg/ml of the substrate and 0.1 M glucose in 50 mM NaAc, pH 5.0. The reaction was stopped by adding two volumes of 1 M Na₂CO₃, and the absorbance of the mixture was measured at the wavelength of 370 nm. The production of the endoglucanase EGI of the HAC1 disruptant was about 50% of the level produced by the parental strain (FIG. 5).

EXAMPLE 3 Cloning and Sequence of the Aspergillus nidulans hacA and Trichoderma reesei HAC1 Genes

[0113] A homology search was performed against a public database (http://bioinfo.okstate.edu/pipeonline.db/anesguery.html )containing Aspergillus nidulans EST (expressed sequence tag) sequences with the yeast HAC1 protein sequence using the program BLAST (Altschul et al., 1990, J. Mol. Biol. 215,403-410). The search identified one EST cDNA clone (c7a10a1.r1) which has homology to yeast HAC1p at the DNA binding domain. However, another region of the same cDNA clone, designated as EST c7a10a1.f1 in the database, had no obvious similarity with HAC1 and there was no annotation within the database to indicate similarity between the ESTs and HAC1. Therefore, it was unclear if the A. nidulans cDNA clone encoded a functional homolog of HAC1 or a different protein having a version of a DNA-binding motif. The region corresponding to the c7a10a1 EST cDNA was amplified by PCR from A. nidulans genomic DNA isolated with methods known in the art. The sequences of the ends of the EST cDNA clone found from the databese were used to design the the 5′ end primer (5′ GCC ATC CTT GGT GAC TGA GCC 3′) (SEQ ID No. 24) and 3′ end primer (5′ CAA TTG CTC GCT CTT ACA TTG AAT 3′) (SEQ ID No. 25). The PCR reaction was performed as described in Example 2. The PCR product of 1.6 kb in length was run in an 0.8% agarose gel, purified from the gel with the Qiaquick gel extraction kit (Qiagen) and cloned into the pGEM-AT vector (Promega) with methods known in the art. The Whole fragment was sequenced from the resulting plasmid using internal oligonucleotide primers.

[0114] To isolate the HAC1 cDNA from Trichoderma reesei, the proper hybridisation temperature for cDNA library screening were determined by genomic Southern hybridization with the genomic hacA fragment cloned from A. nidulans as a probe. The probe fragment was labelled with ³²P-dCTP using the Random primed DNA labelling kit (Boehringer Mannheim) as instructed by the manufacturer. The hybridization was performed as described (Sambrook et al., 1989, in Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) at 48° C., 50° C., 55° C. and 60° C. in a hybridization mixture containing 6×SSC, 5× Denhardt's, 0.5% SDS, 100 μg/ml herring sperm DNA (SSC is 0.15 M NaCl, 0.015 M Na-citrate, pH 7.0, 50× Denhardt's is 1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin). The filters were washed for 10 minutes at room temperature with 2×SSC, 0.1% SDS and for 30 minutes at the hybridization temperature with the same solution. The T. reesei cDNA library constructed into the vector λZAP (Stratagene, Stalbrand et al., 1995, Appl. Environ. Microbiol. 61, 1090-1097) was plated with the appropriate E. coli host strain, and the λ-DNA was lifted onto nitrocellulose filters (Schleicher & Schull) as instructed by the manufacturer. Hybridization of the filters was done for 18 hours at 55° C. in the same hybridization mixture as the Southern hybridization. The filters with λ-DNA were washed for 10 minutes at room temperature with 2×SSC, 0.1% SDS and for 30 minutes at 55° C. with the same solution. The λ-clones hybridizing with the probe were excised into pBluescript plasmids containing the cDNA inserts as instructed (Stratagene). The cDNA clone carrying the largest insert (in the plasmid pMS119, FIG. 6) was chosen for sequencing, and the whole sequence of its insert was determined with the help of internal sequencing primers. The genomic copy of the T. reesei gene was isolated by hybridization of a genomic λ-library in the vector λEMBL3 (Kaiser and Murray, 1985, in DNA Cloning: a Practical Approach, pp. 1-47, ed. Glover, IRL Press, Oxford). The library was plated with the appropriate E. coli host strain and λ-DNA was lifted onto nitrocellulose filters (Schleicher & Schull) as instructed by the manufacturer. The filters were hybridized at 42° C. over night in a hybridization mixture containing 50% formamide, 5× Denhardt's, 5×SSPE, 0.1% SDS, 100 μg/ml herring sperm DNA and 1 μg/ml polyA-DNA (SSPE is 0.18 M NaCl, 1 mM EDTA, 10 mM NaH₂PO₄, pH 7.7). The filters were washed for 10 minutes at room temperature with 2×SSC, 0.1% SDS and 30 minutes at 65° C. in 0.1×SSC, 0.1% SDS. λ-DNA was isolated from clones hybridizing with the probe with a described method (Sambrook et al., 1989, in Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and the genomic region corresponding to the HAC1 cDNA was sequenced from this DNA with internal sequencing primers.

[0115] The sequences of the Trichoderma reesei HAC1 and Aspergillus nidulans hacA genes are shown in FIGS. 7 and 8, respectively. Comparison of the genomic and cDNA sequences from both fungi (the cDNA sequence of hacA available in the EST database) reveals a conventional intron with consensus border sequences at a conserved position in both of the genes. A second intron of 20 bp is found in the T. reesei HAC1 gene. This intron does not have the consensus 5′ border sequence (GT). The sequence around the 5′ end of this intron is predicted to have a strong tendency to form a RNA secondary structure called hairpin loop. The area between the stems of the loop has a sequence very similar to the consensus sequence found at both of the intron borders of the unconventional intron of 252 bp found in yeast HAC1 (FIG. 9, Gonzalez et al., 1999, EMBO. J. 18, 3119-3132). When the yeast UPR pathway is induced, the IRE1 protein cleaves the HAC1 mRNA at these intron borders, and thus initiates the splicing of the intron and formation of an active HAC1 protein. In the Aspergillus nidulans hacA gene there is a sequence almost identical to the hairpin-unconventional intron region of T. reesei HAC1.

[0116] It has been shown by RT-PCR studies that the 20 bp intron is removed from the T. reesei HAC1 and A. nidulans hacA mRNAs upon UPR induction (Example 4). The 250 bp intron in yeast HAC1 prevents translation of the mRNA probably by forming a specific secondary structure (Chapman and Walther, 1998, Curr. Biol. 7, 850-859). The 20 bp intron in the HAC1/hacA genes of filamentous fungi can not form such secondary structures, and thus the activation mechanism of these genes is different from yeast HAC1. The T. reesei HAC1 cDNA encodes an open reading frame of 451 amino acids and the A. nidulans hacA a protein of 350 amino acids, when the 20 bp introns have been removed from the both sequences. The putative T. reesei and A. nidulans HAC1/A proteins have an identity of 37.4% with each other and both have a DNA binding domain conserved with yeast HAC1 protein (FIG. 10). The yeast HAC1 binding site has approximately 64% similarity and 53% identity to the binding site of T. reesei, and approximately 65% similarity and 56% identity to the binding site of A. nidulans. At other regions there is no detectable homology between yeast HAC1 p and the HAC1 of T. reesei or the HACA of A. nidulans. The HAC1 cDNA clone sequenced from T. reesei has a 5′ flanking region of 471 bp, containing two short open reading frames encoding 17 and 2 amino acids. The 5′ flanking region sequenced from A. nidulans hacA is 187 bp in length, containing one upstream open reading frame of 7 amino acids.

EXAMPLE 4 Demonstration of Truncation at the 5′ End and Splicing of the 20 bp Intron of T. reesei and A. nidulans HAC1/hacA mRNA Upon UPR Induction

[0117] When the UPR pathway is induced in yeast, the unconventional intron of the HAC1 gene is spliced and thus the length of the HAC1 mRNA is reduced by 250 bp (Cox and Walter, 1996, Cell 87, 391-404). It was studied if UPR induction affects the length of the HAC1/hacA mRNA in T. reesei and A. nidulans. The T. reesei strain RutC-30 (Montenecourt and Eveleigh, 1979, Adv. Chem. Ser. 181, 289-301) was grown in a shake flask in a Trichoderma minimal medium (Penttilä et al., 1987, Gene 61,155-164) with 2% lactose as the carbon source. Growth was performed for 60 hours at 28° C. and 200 RPM, and the mycelium was diluted 1:10 into the same medium and grown for additional 21 hours. The culture was subsequently divided into two halves, and one half of it was treated with 10 mM dithiothreitol (DTT) to induce the UPR pathway (Saloheimo et al., 1999, Mol. Gen. Genet. 262, 35-45). Mycelial samples were collected from the culture treated with DTT and the untreated control culture before DTT addition and 30, 60, 90, 120 240 and 360 minutes after the addition of DTT. Total RNA was isolated from the samples with the TRIzol reagent (Gibco-BRL) according to manufacturer's protocols. RNA samples of 5 μg were treated with glyoxal and run in a 1% agarose gel in 10 mM Na-phosphate buffer, pH 7.0. Capillary blotting onto a Hybond-N nylon filter (Amersham) was done as instructed by the manufacturer. The full-length HAC1 cDNA that was used as a probe was labelled as described in Example 3. Hybridization was performed for 18 hours at 42° C. in 50% formamide, 10% dextran sulphate, 1% SDS, 1 M NaCl and 125 μg/ml of herring sperm DNA. The filter was washed in 5×SSPE for 15 minutes at 42° C., in 1×SSPE, 0.1% SDS for 30 minutes at 42° C. and in 0.1×SSPE, 0.1% SDS for 30 minutes at room temperature. The results (FIG. 11) show that the length of the HAC1 mRNA does not change in the control samples not treated with DTT. In the samples treated with DTT a shorter mRNA of about 2.2 kb appears in addition to the 2.5 kb mRNA observed in the control samples. The full-length HAC1 cDNA probe was removed from the Northern filter by incubating it in 0.1% SDS at 100° C. for 10 minutes. The filter was then hybridized with a probe containing a 160 bp sequence from the 5′ flanking region of the HAC1 gene. This probe was made by PCR from the plasmid pMS119 (FIG. 6) with the T3 primer (5′ MT TAA CCC TCA CTA AAG GG 3′) (SEQ ID No. 26) binding to the pBluescript vector as the forward PCR primer and the oligonucleotide 5′ TGG TTG ATG ACG ACG ATGCGA ACA GTC ATG ACA GGC AAC G 3′ (SEQ ID No. 27) as the reverse primer. The PCR reaction was performed as described in Example 2. The probe preparation was done as in Example 3. The Northern hybridisation with the short fragment was done as described above for the full-length HAC1 cDNA probe. The short probe fragment derived from the 5′ flanking region of the HAC1 cDNA hybridized with the full-length HAC1 mRNA of 2.5 kb but not with the 2.2 kb mRNA that appears when UPR is induced by DTT, indicating that the 5′ end is the segment absent in the 2.2 kb mRNA. It has previously been shown that the T. reesei pdi1 gene is controlled by the UPR (Saloheimo et al., 1999, Mol. Gen Genet. 262, 35-45). To show that the UPR is induced in this experiment with DTT, the filter was probed with the pdi1 and gpd1 probes. The pdi1 mRNA becomes more abundant in the mycelium treated with DTT, whereas the gpd1 mRNA remains at an almost constant level.

[0118] To analyse more closely the change that occurs in the T. reesei HAC1 mRNA upon UPR pathway induction, the mRNA populations in induced and uninduced conditions were studied by rapid amplification of cDNA ends by PCR (RACE-PCR). PolyA+RNA was isolated from total RNA samples derived from a DTT-treated and an untreated control mycelia, using the OligoTex mRNA isolation kit (Qiagen) as instructed by the manufacturer. The Marathon cDNA amplification kit (Clontech) was used in the RACE-PCR procedure according to manufacturer's protocols. The HAC1-specific oligonucleotide used in the reaction was 5′ GGG AGA CGA CTG CTG GAA CGC CAT 3′ (SEQ ID No. 28). It binds 500 bp downstream from the 5′ end of the full-length HAC1 cDNA. The isolated mRNA was used in synthesis of double-stranded cDNA. An oligonucleotide adapter was ligated to the ends of the cDNA, and the 5′ ends of the HAC1 cDNAs in each sample were amplified by PCR with the HAC1-specific primer and a primer supplied in the kit that binds to the ligated adapter. The PCR program consisted of 5 cycles with denaturation at 94° C. for 5 seconds and synthesis at 72° C. for 3 minutes followed by 5 cycles with denaturation at 94° C. for 5 seconds and synthesis at 70° C. for 3 minutes and 25 cycles with denaturation at 94° C. for 5 seconds and synthesis at 68° C. for 3 minutes. The PCR products were analysed in a 1% agarose gel. A fragment of the expected size (about 550 bp, including the 5′ flanking region of the HAC1 gene and the adapter ligated to the end), corresponding to the 2.5 kb mRNA, was obtained from the control sample derived from mycelia not treated with DTT. A second fragment of about 250 bp, corresponding to the 2.2 kb mRNA size, was obtained in the PCR from the sample treated with DTT in addition to the one observed in the control sample. The 550 bp fragment of the control sample and the 250 bp fragment from the DTT-treated sample were isolated from the agarose gel with the Qiaquick gel extraction kit (Qiagen) as instructed by the manufacturer, and cloned into the pCR2.1-TOPO vector with the TOPO TA cloning kit (Invitrogen) as instructed by the manufacturer. Two independent clones derived from the control RNA and carrying the 550 bp insert were sequenced. They had their 5′ end 8 bp and 16 bp downstream from the 5′ end of the full-length cDNA (nucleotides 8 and 16 in the sequence in FIG. 7) and the sequence continued until the end of the HAC1-specific primer as in FIG. 7. Seven independent clones derived from the DTT-treated mycelium and carrying 250 bp inserts were sequenced. The 5′ ends of these fragments were each at different positions between nucleotides 254 and 336 in the sequence in FIG. 7, and in each case the sequence continued until the end of the HAC1-specific primer as in FIG. 7. This further confirms that the 5′ end of the T. reesei HAC1 mRNA is absent when the UPR pathway is induced by DTT. The upstream open reading frame (uORF) of 17 amino acids is in the region that is left out from the mRNA. Thus this uORF could be involved in the regulation, preventing translation initiation at the correct start codon and formation of the HACI protein.

[0119] The splicing of the 20 bp intron from the T. reesei HAC1 mRNA upon UPR induction was studied by reverse transcriptase-PCR (RT-PCR). The mRNA samples used in RACE-PCR (previous paragraph), one treated with 10 mM DTT and the other not treated, were subjected to first strand cDNA synthesis with the Riboclone cDNA synthesis system (Promega) according to manufacturer's instructions. A fragment of about 500 bp in length, covering the region with the 20 bp intron in the HAC1 gene, was amplified by PCR from the synthesized cDNA using the forward primer 5′ CCC CGA GCA GTC CTT GAT GG 3′ (SEQ ID No. 29) and the reverse primer 5′ GTC GTT GAT GTC GAA GT 3′ (SEQ ID No. 30). The PCR program consisted of denaturation at 94° C. for 2 minutes followed by 30 cycles with denaturation at 94° C. for 45 seconds, annealing at 50° C. for 30 seconds and synthesis at 72° C. for 1 minute. A final synthesis step of 5 minutes at 72° C. was performed. The DNA fragments obtained in the PCR were cloned into the pCR2.1 vector with the TOPO TA cloning kit (Invitrogen) as instructed by the manufacturer. Ten fragments derived from both the DTT-treated sample and the nontreated control sample were sequenced. Nine out of the ten fragments from control sample had the intron unspliced. Only two out of the ten fragments from the DTT-treated sample had the intron unspliced, showing that splicing of the intron occurs upon UPR induction by DTT.

[0120] To examine whether the 5′ flanking region and the 20 bp intron are removed from the Aspergillus nidulans hacA mRNA upon UPR induction similarly to the T. reesei HAC1 mRNA, Northern hybridisation and RT-PCR experiments were carried out. The A. nidulans strain FGSC A26 was grown for three days in shake flasks in a medium containing 3% glucose, 2.5% corn steep liquor, 15 g/l KH₂PO₄, 5 g/l (NH₄)₂SO₄, 5 mg/l FeSO, 1.6 mg/l MnSO₄, 1.4 mg/l ZnSO₄, 3.7 mg/l CoCl₂, pH 6.8. The culture was divided into two aliquots, and one aliquot was treated with 20 mM DTT and the other served as a control. Samples were withdrawn from both aliquots at 0, 30, 60, 120 and 240 minutes after the DTT addition. The mycelium was washed with 0.9% NaCl and stored frozen at −70° C. Total RNA was isolated from the mycelia with the Trizol reagent (Gibco-BRL) as instructed by the manufacturer. Agarose gel electrophoresis, Northern blotting and hybridization of the RNA samples was performed as described in the first paragraph of this example. The Northern was first probed with the full-length hacA genomic fragment shown in FIG. 8. The probe hybridizes with a single 1.7 kb mRNA band in samples not treated with DTT. In the samples treated with DTT for 120 and 240 minutes, an additional band of about 1.55 kb is detected, showing that the hacA mRNA is truncated upon UPR induction (FIG. 12). The Northern was then probed with a short probe derived from the 5′ end of the hacA gene. The probe fragment was made by PCR from the pGEM-AT vector carrying the hacA gene (Example 3) with the T7 primer (5′ GTA ATA CGA CTC ACT ATA GGG C 3′) (SEQ ID No. 31) as the forward primer and hacA-specific oligonucleotide 5′ TTA GGA CAG AGG CCA CGG TGT 3′ (SEQ ID No. 32) as the reverse primer. The PCR reaction was performed as described in the previous paragraph. The 5′ end probe has the first 90 bp of the sequence in FIG. 8. The short 5′ end probe hybridizes only with the 1.7 kb mRNA, showing that the hacA mRNA is truncated from the 5′ end when the UPR pathway is induced.

[0121] To test if the 20 bp intron is removed from the A. nidulans hacA gene when UPR is induced by DTT, RT-PCR was performed. The total RNA samples isolated from mycelia treated with 20 mM DTT for 240 minutes and from control mycelia were subjected to RT-PCR reactions with the Robust RT-PCR kit (Finnzymes, Finland) as instructed by the manufacturer, using the forward primer 5′ CCC ATC CTT GGT GAC TGA GCC 3′ (SEQ ID No. 33) and the reverse primer 5′ MG AGT CGG TGT CAG AGT TGG 3′ (SEQ ID No. 34). The DNA fragment of about 400 bp obtained in the PCR was cloned into the pCR2.1-TOPO vector with the TOPO TA cloning kit (Invitrogen) as instructed by the manufacturer. Twelve of the cloned fragments derived from DTT-treated and ten from control mycelia were sequenced. None of the fragments derived from the control mycelia had the intron spliced. Three of the fragments derived from the DTT-treated mycelia had the intron spliced.

EXAMPLE 5 Complementation of Yeast HAC1 and IRE1 Disruptions by Different Forms of the T. reesei HAC1 cDNA

[0122] The S. cerevisiae IRE1 gene was disrupted in the same way as the HAC1 gene (described in Example 2). A fragment where a G418 resistance cassette is flanked by sequences from the 5′ and 3′ ends of the IRE1 open reading frame was made by PCR. The forward primer 5′ ATT AAT ATT TTA GCA CTT TGA AAA ATG CGT CTA CTT CGA AGA AAC ATG CTT GCC TCG TCC CCG CCG GGT CAC 3′ (SEQ ID No. 35) and the reverse primer 5′ AAG CAG AGG GGC ATG AAC ATG TTA TGA ATA CAA AAA TTC ACG TAA MT GTC GAC ACT GGA TGG CGG CGT TAG TAT 3′ (SEQ ID No. 36) were used in the PCR reaction. The PCR reaction, yeast transformation, and selection and analysis of the disruptants were performed as described in Example 2 for HAC1 disruption.

[0123] To express different forms of the T. reesei HAC1 gene in the yeast HAC1 and IRE1 disruptants, four expression constructs were made into the multicopy expression vector pAJ401 (Saloheimo et al., 1994, Mol. Microbiol. 13, 11-21)with the URA3 marker gene and yeast PGK1 promoter and terminator to drive the expression. One of them has the HAC1 cDNA with the intact 5′ flanking region and does not have the 20 bp intron. This plasmid, pMS131 (FIG. 13), was made by releasing the HAC1 cDNA from pMS119, which is the pBluescript vector (Stratagene) carrying the full-length cDNA, with EcoRI and Asp718 digestion, filling in the ends of the fragment with Klenow polymerase and ligating it to the EcoRI restriction site of pAJ401 with methods known in the art. The second construct has the T. reesei HAC1 cDNA truncated at the 5′ end but does not have the 20 bp intron. The truncated HAC1 cDNA fragment was made by PCR from the plasmid pMS119 (FIG. 6) with the forward primer 5′ CCG CAA CAC GAC ACG GCA GGC AAC 3′ (SEQ ID No. 37) and reverse primer 5′ CTA GGT AGA CGT TGT ATT TTG 3′ (SEQ ID No. 38). The PCR reaction was carried out as described in Example 2. The PCR product was run in a 0.8% agarose gel and purified from it with the Qiaquick gel extraction kit (Qiagen). The fragment was cloned into the EcoRV restriction site of the pZERO vector using the Zero Background Cloning kit (Invitrogen) according to manufacturer's protocols. The fragment was released from this vector with BamHI digestion and cloned between the EcoRI and XhoI restriction sites of the pAJ401 vector with methods known in the art. The resulting plasmid was named pMS132 (FIG. 14). The third and fourth expression plasmids have the 20 bp intron added to the HAC1 cDNA forms either with or without the 5′ flanking region. These plasmids were constructed by replacing a HpaI-KspI fragment of about 800 bp in pMS131 and pMS132with a corresponding HpaI-KspI fragment from a cDNA which has the 20 bp intron, isolated from the cDNA library in λZAP together with the cDNA in the plasmid pMS119 (Example 3).

[0124] To test for complementation, the four expression plasmids and the vector pAJ401 alone were transformed into the yeast HAC1 and IRE1 disruptants as described (Gietz et al., 1992, Nucl. Acids Res. 20, 1425). Four transformants from each of the transformations were streaked on SC-Ura plates (Sherman, 1991, Meth. Enzymol. 194, 3-21) and grown at 30° C. for three days. The plates were then replicated onto mineral medium plates (Verduyn et al., 1992, Yeast 8, 501-517) with inositol and on plates without inositol. These plates were incubated at 30° C. for three days and the streaks growing on them were replicated again onto the same plates. After growth of five days the inositol dependence of the transformants was evaluated (FIG. 15). Both pMS131 (HAC1 cDNA with 5′ flanking region and without intron) and pMS132 (without 5′ flanking region, without intron) could restore the ability of both the HAC1 and IRE1 disruptants to grow without inositol. Thus the T. reesei HAC1 encodes the functional homolog of the yeast HAC1 gene. When the 20 bp intron is added to pMS131, no complementation is obtained. When the intron is added to pMS132, the yeast disruptants grow very slowly without inositol. Thus the 20 bp intron weakens the ability of the T. reesei HAC1 gene to complement the yeast HAC1 and IRE1 disruptions.

EXAMPLE 6 Binding of the T. reesei HAC1 Protein to UPR Elements of the pdi1 and bip1 Promoters

[0125] A fragment of the T. reesei HACI protein containing the putative DNA binding domain and leucine zipper region was produced in E. coli as a fusion protein with the E. coli maltose-binding protein maIE. A DNA fragment encoding this part of the HACI protein was prepared by PCR from the HAC1 cDNA with the oligonucleotide primers 5′ TCG AAC GGA TCC GAA AAG AAG CCC GTC AAG AAG AGG 3′ (forward primer) (SEQ ID No. 39) and 5′ ATC GCA GGA TCC CTA GGT TTG GCC ATC CCG CGA GCC AAA 3′ (reverse primer) (SEQ ID No. 40). The PCR reaction was performed as in Example 2. The PCR product of 360 bp was run in an 0.8% agarose gel and purified from the gel with the Qiaquick gel extraction kit (Qiagen). The fragment was digested with BamHI at the restriction sites included in the PCR primers and cloned into the BamHI restriction site of the vector pMAL-p2X (New England Biolabs) with methods known in the art. The HACI-maIE protein was produced in E. coli and purified by amylose affinity chromatography using the pMAL Protein Fusion and Purification System (New England Biolabs) as recommended by the manufacturer. The E. coli cells were grown up to OD600 0.5 at 37° C., IPTG was added to the concentration of 0.3 mM, and production was carried out for 3 hour at 24° C. The HACI-maIE fusion protein with the expected apparent molecular weight was purified.

[0126] The oligonucleotides used in binding reactions were annealed in the concentration of 100 mg/ml in 50 mM Tris, pH 8.0, 10 mM MgCl₂, 1 mM spermidine and 5 mM DTT by heating them at 65° C. for 10 minutes and letting them cool down to room temperature during 2 hours. The oligonucleotides were labelled by incubating 100 ng of the annealed oligonucleotide in 10 mM Tris, pH 8.0, 5 mM MgCl₂ with 20 μCi of ³²P-dCTP and 2.5 U Klenow polymerase (Boehringer Mannheim) at 37° C. for 30 minutes. The binding reactions between the oligonucleotides having the putative UPR elements and the proteins were carried out with 0.5-2 μg of the HACI-malE fusion protein or 2 μg of the maIE protein and 1 ng of the annealed and labelled oligonucleotide in a mixture containing 20 mM HEPES, pH 6.9, 50 mM KCl, 10 mM MgCl₂, 0.25 mM EDTA, 0.5 mM DTT, 2% Ficoll, 5% glycerol and 100 μg/ml poly(dIdC) DNA. The competing oligonucleotides were used in 20-200 times excess of the labelled oligonucleotide. The binding eaction mixtures were incubated for 30 minutes at 25° C. and run in a 5% polyacrylamide gel with 10% glycerol in 12.5 mM Tris-borate, pH 8.3, 0.6 mM EDTA for three hours. The gel was dried on a filter paper and exposed onto an X-ray film.

[0127] The following oligonucleotides carrying the putative UPR elements of the pdi1 and bip1 promoters were used in the binding reactions (only the leading strand is given, the UPREs are given in bold):

[0128] pdiUPREI+II, containing both of the putative UPR elements of the pdi1 promoter (Saloheimo et al. 1999, Mol. Gen. Genet. 262, 35-45).

[0129] 5′ CGG CTG AAC CAG CGC GGC AGC CAG ATG TGG CCA AAG GG 3′ (SEQ ID No. 41)

[0130] pdiUPREI, containing the UPREI of the pdi1 promoter in a random context 5′ GGT ACC TGC TAA CCA GCG CGG CAT GAT TCA AC 3′ (SEQ ID No. 42)

[0131] pdiUPREII, containing the UPREII of the pdi1 promoter in a random context 5′ GGA TCT TGC ATA GCC AGA TGT GGC CTC GAT TGA CT 3′ (SEQ ID No. 43)

[0132] bipUPREI, containing the UPREI of the bip1 promoter (unpublished results) 5′ GGA TTA GM AAC GCC AAC GTG TCC ATA ACG GTC 3′ (SEQ ID No. 44)

[0133] bipUPREII, containing the UPREII of the bip1 promoter, the element is in a reverse orientation in the promoter (unpublished results) 5′ GGG CGT GGA GM GCG AGA AGT GGC CTC TTC TTC TCC 3′ (SEQ ID No. 45)

[0134] The results (FIG. 16) show that the HACI-maIE fusion protein binds to the putative UPR element area found from the pdi1 promoter whereas the maIE protein alone does not show any binding. The binding of the fusion protein is specific, since it is competed by an excess of unlabelled oligonucleotide. The fusion protein binds specifically also to the oligonucleotide pdiUPREII and not at all to pdiUPREI, and this indicates that the functional UPR element of the pdi1 promoter is UPREII. The HACI-maIE fusion protein also binds specifically to both of the putative UPR elements found in the bip1 promoter. Alignment of the three T. reesei UPR element shows that the consensus sequence for binding is GC(C/G)A(G/A)N₁₋₂GTG(G/T)C (FIG. 16) (SEQ ID No. 46).

EXAMPLE 7 Expression in Yeast of the Trichoderma HAC1 cDNA Without Its 20 bp Intron and Truncated at the 5′ End

[0135] The T. reesei HAC1 cDNA was expressed without its 5′ flanking region and without the 20 bp intron from the plasmid pMS132 (FIG. 14). This plasmid and the control plasmid pAJ401 were transformed with a described method (Gietz et al., 1992, Nucleic Acids Res. 20, 1425) into the yeast strain producing Bacillus amyloliquefaciens α-amylase described in Example 1,. Two strains carrying pMS132 and two strains with pAJ401 were grown for six days in shake flasks (250 RPM, 30° C.) in SC-Ura medium (Sherman, 1991, Meth. Enzymol. 194, 3-21) buffered to pH 6.0 with 2% succinic acid and growth and amylase production were assayed as described in Example 1. Cell samples were withdrawn from the culture for Northern analysis. The α-amylase production of the pMS132 transformants calculated per biomass was higher than that of the pAJ401 transformants from day 3 until the end of the cultivation (FIG. 17). Growth of the pMS132 strains was slower than the growth of the control plasmid strains. Four pMS132 transformants and four pAJ401 transformants were grown in shake flasks (250 RPM, 30° C.) in SC-Ura with 2% sucrose as the carbon source, and invertase activity produced by the cells was assayed as described in Example 1. More invertase was produced by the pMS132 transformants than by the pAJ401 transformants (FIG. 18).

[0136] To show that the truncated T. reesei HAC1 cDNA is beneficial for α-amylase and invertase production by inducing the UPR pathway of yeast, Northern analysis was performed on the cell samples withdrawn from the cultures of pMS132 and pAJ401 transformants. Total RNA was isolated from the cells collected after 1, 2 and 3 days of growth with the RNeasy RNA extraction kit (Qiagen) as instructed by the manufacturer. The yeast KAR2 gene is under the UPR pathway control (Cox and Walter, 1996, Cell 87, 391-404), and therefore the Northern filter was probed with a fragment derived from KAR2. This fragment was produced by PCR from yeast chromosomal DNA with the oligonucleotide primers 5′ GTG GTA ATA TTA CCT TTA CAG 3′ (SEQ ID No. 47) (forward primer) and 5′ CAA TTT CM TAC GGG TGG AC 3′ (reverse primer) (SEQ ID No. 48). A fragment from the yeast TDH1 gene encoding glyceraldehyde phosphate dehydrogenase was used as a control probe, since this gene is expressed constitutively and is not expected to be affected by UPR. The TDH1 probe fragment was made from yeast chromosomal DNA by PCR with the oligonucleotide primers 5′ TGT CAT CAC TGC TCC ATC TT 3′ (forward primer) (SEQ ID No. 49) and 5′ TTA AGC CTT GGC AAC ATA TT 3′ (reverse primer) (SEQ ID No. 50). The PCR reaction was done as in Example 2 and the probes were prepared as described in Example 3. Northern blotting and hybridization were performed from the RNA samples as described in Example 4. The filter was exposed to the screen of the phosphoimager SI (Molecular Dynamics), and the signal intensities were quantified with the phosphoimager. The KAR2 signal intensities were normalized with reference to the TDH1 signal intensities. The results (FIG. 19) show that the KAR2 mRNA abundance is 2-4-fold higher in the pMS132 transformants than in the pAJ401 transformants in all the timepoints.

EXAMPLE 8 Expression in Trichoderma reesei of the HAC1 Gene Without Its 20 bp Intron and Truncated at the 5′ End

[0137] To induce the UPR pathway constitutively, a form of the T. reesei HAC1 cDNA that is truncated at its 5′ flanking region and does not have the 20 bp intron was expressed in T. reesei. The form of the HAC1 cDNA that was present in pMS132 was expressed in yeast as described in Example 5 was cloned with methods known in the art into the NcoI restriction site of the vector pAN52-NotI, between the gpdA promoter and trpC terminator of Aspergillus nidulans. The hygromycin resistance cassette consisting of the A. nidulans gpdA promoter and trpC terminator and the E. coli hygromycin resistance gene was subsequently cloned into the NotI restriction site of the pAN52-NotI carrying the HAC1 cDNA fragment. The resulting plasmid, named pMS136 (FIG. 20), was transformed into T. reesei strain P37PΔCBHIpTEX-CHY22 as described (Penttilä et al., 1987, Gene 61, 155-164). Strain P37PΔCBHIpTEX-CHY22 was constructed by transformation of strain P37PΔCBHIPyr-26 (U.S. Pat. No. 5,874,276) with a version of the expression vector of pTEX-CHY. Vector pTEX-CHY is a derivative of pTEX in which the coding region for the T. reesei cellobiohydrolase I (CBHI) signal sequence, catalytic core and linker region (amino acids 1-476 of CBHI, Shoemaker, et al., 1983, Bio/Technology, 1:691-696) fused to the coding region of bovine prochymosin B (Harris et al., Nucleic Acids Research, 10:2177-2187. was inserted between the cbh1 promoter and terminator region by methods known in the art. Selection of the P37PΔCBHIpTEX-CHY22 transformants with pMS136 was performed on media with 100 μg/ml hygromycin. To obtain uninuclear transformant clones, the transformants were sporulated and single spores were plated on the selective medium with hygromycin. Purified transformants and the parental strain used in the transformation were grown in shake flasks (28° C., 200 RPM) in Trichoderma minimal medium (Penttilä at al., 1987, Gene 61, 155-164) supplemented with 3% whey and 0.2% peptone. Mycelial samples were collected from the cultures on the third, fifth and sixth cultivation days. Total RNA was isolated from the mycelia with the TRIzol reagent (Gibco-BRL) as instructed by the manufacturer. Northern blotting and hybridization were performed to the RNA samples as described in Example 4. The Northern filter was first probed with the full-length HAC1 cDNA, and an mRNA derived from the expression construct which is about 2.0 kb in length can be observed in two of the transformants in addition to the 2.5 kb band that is derived from the native HAC1 gene (FIG. 21). The HAC1 probe was removed from the Northern filter by incubating it in 0.1% SDS at 100° C. for 10 minutes. The filter was subsequently probed with the T. reesei pdi1, bip1 and gpd1 probes. Pdi1 encodes the protein disulphide isomerase and has been shown to be regulated by the UPR pathway (Saloheimo et al., 1999, Mol. Gen. Genet. 262, 35-45). Bip1 (unpublished) encodes the T. reesei homologue of the ER-specific chaperone protein Bip. The gpd1 gene encodes glyceraldehyde phosphate dehydrogenase and was used as the constitutive control probe. After hybridization the filter was exposed to the screen of the Phosphoimager SI (Molecular Dynamics) and the signals were quanified with the phosphoimager. The pdi1 and bip1 signals were normalized with respect to the gpd1 signals. The results show that in the two transformants which express the truncated HAC1 mRNA the pdi1 mRNA level is 4- and 7-fold higher than in the parental strain on the third culture day (FIG. 21). This indicates that the UPR pathway can be induced constitutively in Trichoderma reesei by the expression of HAC1 gene without its 20 bp intron and 5′ flanking region

EXAMPLE 9 The Effect of a T. reesei HAC1 Mutation on Heterologous Protein Production

[0138] A Trichoderma reesei strain where the HAC1 gene is mutated was unexpectedly generated during the transformation of the plasmid pMS136 into the strain producing CBHI-chymosin fusion protein (Example 7). When analysing the transformants by Northern hybridization it was noticed that one of the transformants (number 31) produced several forms of the HAC1 mRNA that are considerably shorter than 2 kb (FIG. 21 lanes 4, 8 and 12). On the fifth and sixth day of the culture as described in Example 7 the unfolded protein response is induced in the parental strain of the transformation, presumably by the production of the heterologous protein chymosin. This is seen in the Northern analysis as appearance of a HAC1 mRNA of about 2.2 kb (truncated at the 5′ flanking region) and as the induction of the pdi1 mRNA on days 5 and 6 (FIG. 21). It has previously been shown that the production of antibody Fab fragments induces the pdi1 gene (Saloheimo et al., 1999, Mol. Gen. Genet. 262, 35-45). In the transformant number 31 the 2.2 kb HAC1 mRNA and the induction of the pdi1 and bip1 mRNAs are not detected, suggesting that the HAC1 gene of this strain is functionally impaired. To further verify this, a DTT treatment experiment of the transformant number 31 was carried out. It was grown in shake flasks (28° C., 200 RPM) in the Trichoderma minimal medium (Penttilä at al., 1987, Gene 61, 155-164) with 3% whey and 0.2% peptone for three days. The culture was divided into two aliquots and one of them was treated with 10 mM dithiothreitol (DTT) and the other served as the control. Samples were taken from both aliquots at 0, 30, 60, 120 and 240 minutes after DTT addition. Total RNA was isolated from the mycelia and Northern hybridization was performed as described in Example 7. Hybridization of the Northern with the HAC1 probe reveals that the UPR induction by DTT is severely delayed in the transformant number 31. The HAC1 mRNA of 2.2 kb is detected only 4 hours after DTT addition (FIG. 22) and a 2-fold induction of the pdi1 gene is also apparent in this timepoint. In a wild type strain the 2.2 kb HAC1 mRNA appears and the pdi1 induction takes place after 30 minutes of DTT treatment (Example 4, FIG. 11).

[0139] The chymosin levels produced by the control strain and the transformant number 31 were measured daily from the media of the whey-peptone cultures described in example 7. The measurements were done from two parallel cultures with a milk clotting assay (Cunn-Coleman, et al., 1991, Bio/Technology, 9:976-981. Transformant number 31 produced roughly the same amount of chymosin as the parental strain on days 2 and 3 of the culture. On the later days the chymosin levels in the culture of the mutant strain started declining, whereas the control strain could still increase significantly the chymosin amount in its culture medium (FIG. 23). The difference between the two strains is evident in the late stages of the culture, where the UPR pathway is induced in the parental strain but not in the strain number 31. This suggests that a functional HAC1 gene and induction of the UPR pathway in the late culture stages is needed for efficient production of CBHI-chymosin fusion protein in T. reesei.

EXAMPLE 10 Cloning and Sequences of the Aspergillus nidulans ptcB and Trichoderma reesei ptc2 Genes

[0140] The yeast protein phosphatase encoded by the PTC2 gene has been shown to be involved in the regulation of the UPR pathway (Welihinda et al., 1998, Mol. Cell. Biol. 18, 1967-1977). The IRE1 protein is phosphorylated when the UPR pathway is turned on (Shamu and Walter, 1996, EMBO J. 15:3928-3039), and Ptc2 dephosphorylates IRE1p and regulates the UPR negatively. A BLAST search (Altschul et al., 1990, J. Mol. Biol. 215, 403-410) was made with the yeast Ptc2 sequence against the public database containing Aspergillus nidulans EST cDNA sequences, and the cDNA clone i2c04a1 was found to be homologous, to it within the database. The region corresponding to this cDNA was amplified by PCR from Aspergillus nidulans genomic DNA with the oligonucleotides 5′ TTG AAC AGC AGA TCG TTA CTG 3′ (forward primer) (SEQ ID No. 51) and 5′ TAT AAA GTT CGT CAA TAG TGG 3′ (reverse primer) (SEQ ID No. 52). The PCR reaction was carried out as described in Example 2. The resulting PCR fragment was cloned into the pCR2.1 vector with the TOPO TA cloning kit (Invitrogen) as instructed by the manufacturer. It was sequenced with internal oligonucleotide primers (FIG. 24). The optimal hybridization conditions for isolation of the T. reesei ptc2 cDNA were determined by Southern hybridization of T. reesei genomic DNA with the A. nidulans ptcB fragment as described in Example 3. A T. reesei cDNA library constructed in λZAP (Stratagene, Stalbrandt et al., 1995, Appl. Environ. Microbiol. 61, 1090-1097) was screened by hybridization with the A. nidulans ptcB fragment as described in Example 3. The λ-clones hybridizing with the probe were excised into pBluescript plasmids with the cDNA inserts as instructed (Stratagene), and the clone having the longest insert based on restriction enzyme digestion was chosen for sequencing. The insert of this cDNA clone is 1830 bp in length, encoding an open reading frame of 438 amino acids (FIG. 25). The putative Trichoderma PTCII protein (used interchangeably with PTC2) shows the highest identity among yeast proteins to Ptc2, 48%. It also shares 60% identity with the putative PTC2 protein from Schizosaccharomyces pombe. The ptcB fragment cloned from Aspergillus nidulans is 1264 in length (FIG. 24). Based on homology with other Ptc2 sequences, an intron has been identified in the fragment. The deduced amino acid sequence is 89% identical to T. reesei PTCII protein over an area of 117 amino acids.

EXAMPLE 11 Cloning and Sequences of the Aspergillus nidulans ireA and Trichoderma reesei IRE1 Genes

[0141] A search with the program BLAST (Altschul et al., 1990, J. Mol. Biol. 215, 403-410) was made with the yeast IRE1 protein sequences against the public database containing Aspergillus nidulans EST cDNA sequences. The EST clone v1h01a1 was homologous to yeast IRE1 protein and include such annotation. The region corresponding to this EST cDNA was amplified by PCR from Aspergillus nidulans genomic DNA with the oligonucleotides 5′ CGG AGG CAA GAG TCA TAG ACG 3′ (forward primer) (SEQ ID No. 53) and 5′ CM TAT ATT TCT GAA CCA GTA CG 3′ (reverse primer) (SEQ ID No. 54). The PCR reaction was carried out as described in Example 2. The resulting PCR fragment was cloned into the pCR2.1 vector with the TOPO TA cloning kit (Invitrogen) as instructed by the manufacturer. It was sequenced with internal oligonucleotide primers. The fragment was used as a probe in isolation of the T. reesei IRE1 gene. Optimal hybridization conditions were first determined with Southern hybridization of genomic T. reesei DNA as described in Example 3. A T. reesei genomic library constructed in λEMBL3 (Kaiser and Murray, 1985, in DNA Cloning: a Practical Approach, pp. 1-47, ed. Glover, IRL Press, Oxford) was then plated with the appropriate E. coli host strain and λ-DNA was lifted onto nitrocellulose filters (Schleicher & Schull) as instructed by the manufacturer. The filters were hybridized over night at 50° C. in a mix containing 6×SSC, 5× Denhardt's, 0.5% SDS, 100 μg/ml herring sperm DNA (SSC is 0.15 M NaCl, 0.015 M Na.citrate, pH 7.0, 50× Denhardt's is 1% Ficoll, 1% polyvinylpyrrolidone, 1% bovine serum albumin). The filters were washed for 10 minutes at room temperature with 2×SSC, 0.1% SDS and for 30 minutes at 50° C. with the same solution. λ-DNA was isolated from clones hybridizing with the probe with a described method (Sambrook et al., 1989).

[0142] Most of the protein-coding region of the genomic IRE1 gene was subcloned into pBluescript SK⁻ as 2.1 kb and 2.4 kb BamHI fragments with methods known in the art. These fragments were sequenced with synthetic oligonucleotide primers. The two subclone fragments do not cover the whole open reading frame, and thus the 5′ end of the chromosomal gene was sequenced from DNA isolated from the λ-clone isolated from the genomic library. An IRE1 cDNA was isolated from a T. reesei library constructed in λZAP (Stratagene). The cDNA library was plated with the appropriate E. coil host and lifted onto nitrocellulose filters (Schleicher & Schull) as instructed by the manufacturer. The probe fragment used in the screening was obtained by digesting the 2.4 kb genomic subclone plasmid with BamHI and SmaI. The fragment of about 600 bp was run in a 0.8% agarose gel and isolated from the gel with the Qiaquick gel extraction kit (Qiagen) with manufacturer's instructions. The probe was labelled with ³²P-dCTP with the Random Primed DNA labelling kit (Boehringer Mannheim). The filters were hybridized at 42° C. over night in a hybridization mixture containing 50% formamid, 5×Denhardt's, 5×SSPE, 0.1% SDS, 100 μg/mI herring sperm DNA and 1 μg/ml polyA-DNA (SSPE is 0.18 M NaCl, 1 mM EDTA, 10 mM NaH₂PO₄, pH 7.7). The filters were washed for 10 minutes at room temperature with 2×SSC, 0.1% SDS and for, 30 minutes at. 65° C. in 0.1×SSC, 0.1% SDS. λ-clones giving a hybridization signal were converted into pBluescript plasmids by in vivo-excision as instructed (Stratagene). The T. reesei IRE1 cDNA was sequenced from one of the plasmids with internal oligonucleotide primers.

[0143] The area sequenced from the T. reesei IRE1 gene is about 4.5 kb, and the open reading frame encodes a protein of 1233 amino acids (FIG. 27). Comparison of the genomic and cDNA sequences revealed one intron. The T. reesei IRE1 protein starts with a predicted signal sequence of 25 amino acids. There is a putative transmembrane segment at positions 574-596 of the open reading frame. The N-terminal domain (before the transmembrane segment) presumably facing the lumen of the endoplasmic reticulum has 24% identity and 39% similarity over an area of 377 amino acids with yeast IRE1p. The C-terminal part with the kinase and RNAse domains is 42% identical and 59% similar over an area of 490 amino acids to yeast IRE1p. The cloned A. nidulans ireA fragment is 1570 bp in length (FIG. 26). It encodes the kinase and RNAse domains of the IREA protein. Based in comparison with the yeast and T. reesei IRE1 sequences, an intron is identified in the sequence of the ireA fragment. The deduced A. nidulans IREA amino acid sequence has 52% identity over an area of 507 amino acids to the T. reesei IREI protein.

EXAMPLE 12 Cloning and Constitutive Expression of the Aspergillus niger var. awamori hacA cDNA

[0144] The A. niger var. awamori hacA cDNA was isolated by heterologous hybridisation with the cloned Aspergillus nidulans hacA fragment described in Example 3. A cDNA library constructed from A. niger var. awamori RNA in the plasmid pYES2 (Invitrogen) was plated as E. coli colonies, lifted onto nitrocellulose filters and screened by colony hybridisation as described for the isolation of the T. reesei hac1 cDNA in Example 3. The hybridisation and the final washes were performed at 57° C. Positive colonies were found and examined by restriction analysis and sequencing of the cDNA ends. The longest cDNA was sequenced throughout its length from both strands. It is 1.68 kb long and encodes a protein of 342 amino acids (FIG. 28). The encoded protein has 76% identity with A. nidulans HACA protein and 38% identity with T. reesei HACI protein. The A. niger var. awamori hacA cDNA has an upstream open reading frame encoding 44 amino acids. The region of the cDNA that, according to homology with the T. reesei and A. nidulans hac1/A genes, had a 20 bp intron was sequenced from five of the A. niger var. awamori hacA cDNA clones isolated. One of these clones did not have the 20 bp intron present, showing that the intron can be spliced out as is shown in Example 4 for the 20 bp introns of T. reesei hac1 and A. nidulans hacA genes.

[0145] The UPR-induced form of the A. niger var. awamori hacA cDNA was expressed in A. niger var. awamori strains producing Trametes versicolorlaccase or bovine preprochymosin which were constructed in the following manner. Strains ΔAP3 and ΔAP4 (described in Berka, R. M. et al., 1990, Gene 86:153-162) are equivalent strains which are deleted for the pepA gene (encoding the major extracellular aspartic proteinase) and which have a pyrG null mutation.

[0146] Strain ΔAP3 was transformed with pUCpyrGRG3 to create strain ΔAP3pUCpyrGRG3#11 which produces bovine preprochymosin. This strain secretes and accumulates active chymosin (an aspartic proteinase) in the culture medium. The plasmid, pUCpyrGRG3, consists of the GRG3 expression cassette (encoding the Aspergillus niger glaA promoter, preprochymosin open reading frame and glaA terminator) obtained from pGRG3 (Cullen, D. et al., 1987, Bio/Technology 5:369-376) and the Neurospora crassa pyr4 gene inserted into pUC19. Transformants of strain ΔAP3 with this plasmid were selected on the basis of uridine auxotrophy. Transformants were screened in liquid culture for chymosin production and strain ΔAP3pUCpytGRG3#11 was chosen as the best producer.

[0147] Strain ΔAP4 was transformed with pGPT-LCC1 to create strain ΔAP4:pGPTlaccase which secretes Trametes versicolor laccase 1. The plasmid, pGPT-LCC1, is a derivative of plasmid pGPTpyrG1 (described in Berka, R. M. and Barnett, C. C., 1989, Biotechnol. Adv. 7:127-154) which contains the N. crassa pyr4 gene as fungal selectable marker and the A. niger glaA promoter and A. niger var. awamori glaA terminator region separated by cloning sites. To create pGPT-LCC1 the open reading frame for the Trametes versicolor Icc1 cDNA (Ong, E. et al., 1997, Gene 196:113-119) was inserted between the glaA promoter and terminator regions in pGPTpyrG1. Transformants of strain ΔAP4 with this plasmid were selected on the basis of uridine auxotrophy. Transformants were screened in liquid culture for laccase production and strain ΔAP4:pGPTlaccase was chosen as the best producer.

[0148] For the over expression of hacA, the induced form of the A. niger var. awamori hacA cDNA was first created by deleting the 20 bp intron and truncating the 5′ flanking region by about 150 bp, which omitted the upstream open reading frame. This was done by methods known in the art. The resulting hacA gene fragment was then cloned into an A. niger var. awamori expression vector with methods known in the art. In the final expression construct, pMS152 (FIG. 29), the hacA gene fragment is between the A. niger var. awamori glaA (glucoamylase gene) promoter and terminator. The A. nidulans amdS gene endoding acetamidase was in the plasmid as a selection marker for fungal transformation.

[0149] The hacA overexpression construct (pMS152) was transformed into either A. niger var. awamori strain ΔAP3pUCpyrGRG3#11 or strain ΔAP4:pGPTlaccase. The transformations were performed as described in Penttila et. al., 1987, Gene 61, 155-164. The transformants were selected for the ability to grow on acetamide as the sole nitrogen source. Transformants were passaged three times on selective medium before they were sporulated and single spores were plated on the selective medium.

[0150] For Southern analysis the purified transformants and the parental strains were grown in shake flasks (28° C., 200 rpm) in Clofine special medium (described in WO 98/31821). Mycelial samples for total-DNA isolations were collected on the third cultivation day. The isolations were done with the DNA EASY kit (Invitrogen) according to the manufacturer's instructions. 5 μg of the total DNA was cut with restriction enzyme HindIII and XhoI to obtain a 5.2 kb-fragment from the integrated pMS152 to indicate which transformants have the hacA overexpression cassette and samples were run in 1% agarose gel in 1×TBE-buffer. The treatment of the gels and capillary blotting onto a Hybond-N nylon filter (Amersham) were done as instructed by the manufacturer. A fragment of the A. niger var. awamori hacA cDNA labeled as described in Example 3 was used as a probe in the Southern hybridisation. The filters were hybridised at 42° C. over night in a hybridisation mixture containing 50% formamide, 5× Denhart's, 5×SSPE, 0.1% SDS, 100 μg/ml herring sperm DNA and 1 μg/ml poly (A)-DNA. Filters were washed as described in Example 4. A band of the expected size was obtained from all the transformants that were analysed, but not from the parental strains. This indicated that the obtained transformants were stable and that they contained intact hacA overexpression cassette.

[0151] Eight transformants from the laccase-producing strain and four transformants from the chymosin-producing strain shown to contain the hacA overexpression cassette were cultivated again for Northern analysis and measurement of the enzymatic activities. The pMS152 transformants of the strain producing preprochymosin and the untransformed parental strain (ΔAP3pUCpyrGRG3#11) were cultivated in Clofine special medium in shake flasks (28° C., 200 rpm) in two parallel cultures for six days. Mycelial samples for RNA isolations were taken on the third day of the cultivation. The pMS152 transformants of the strain producing Trametes laccase and the untransformed parental strain (ΔAP4:pGPTlaccase) were cultivated in 8 g/litre Bacto Soytone (Difco), 12 g/litre Tryptone peptone (Difco), 15 g/litre (NH₄)₂SO₄, 12.1 g/litre NaH₂PO₄.H₂O and 3.3 g/litre Na₂HPO₄.7H₂O. After autoclaving 5 ml/litre of 20% MgSO₄ solution, 2 ml/litre of Cu/citrate solution (110 g/litre citrate*H₂O, 125 g/litre CuSO₄.5H₂O), 1 ml/litre Tween 80, 300 ml/litre 50% maltose solution and 200 ml/litre of 100 mg/litre arginine was added to the medium. The cultivations were done in shake flasks (28° C., 200 rpm) in two parallel cultures for ten days. The mycelial samples for RNA isolations were taken on the second day of the cultivation. Total RNA's were isolated from all the mycelial samples using the TRIZOL reagent (Gibco-BRL) as instructed by the manufacturer. RNA samples of 5 μg were treated with glyoxal and run in 1% agarose gel in 10 mM Na-phosphate buffer, pH 7.0. Northern blottings and hybridizations were done as described in Example 4. A fragment of the A. niger var. awamori hacA cDNA labeled as described in Example 3 was used as a probe. An mRNA of the expected size from the hacA overexpression cassette of about 1.6 kb was observed in all the transformants studied in addition to the band of about 1.7 kb that is derived from the native hacA gene and that is also seen in the controls. This indicates that the 5′-truncated and intronless hacA coming from the overexpression cassette is expressed in the transformants.

EXAMPLE 13 The Effect of A. niger var. awamori hacA Overexpression on Heterologous Protein Production

[0152] Samples from the culture supernatants of the pMS152 transformants of the strain producing preprochymosin and the untransformed parental strain (ΔAP3pUCpyrGRG3#11) were taken on the fifth day of cultivation. The chymosin production levels were measured with a milk-clotting assay. The samples were diluted into buffer containing 10 g/litre sodium acetate and 5 ml/litre 1M acetic acid. 200 μl of the diluted sample was added to 5 ml of buffer containing 55 g/500 ml skim milk (Difco) at 30° C. The clotting of the milk was observed visually and the time that the clotting of the milk took was recorded and correlated to a known standard. All the four transformants produced 1.3-2.8 fold more chymosin than the parental strain (FIG. 30).

[0153] Samples from the culture supernatants of the pMS152 transformants of the strain producing Trametes laccase and the untransformed parental strain (ΔAP4:pGPTlaccase) were taken on the fifth and seventh day of the cultivation. The laccase activity measurements were made from the supernatants and the results showed that all the transformants produce more laccase than the parental strain. Laccase activity was measured according to Niku-Paavola et al. (Niku-Paavola M-L, Karhunen E, Salola P, Raunio V (1988) Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem. J. 254: 877-884) using ABTS (Boehringer Mannheim; Mannheim, Germany) as a substrate. The production levels of the transformants in the fifth day samples were 3 to 7.6 fold higher than in the parental strain. On the seventh day of cultivation the transformants produced 2 to 5.4 fold more laccase than the parental strain (FIG. 31).

[0154] These results demonstrate that overexpression of an inducing form of hacA enables production of higher levels of secreted heterologous proteins in A. niger.

[0155] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

1 63 1 2417 DNA Trichoderma reesei 1 cgagaggcca ctctgtcctc ttctgcctga ctcatcactc ctcgacagca tcaccaaggg 60 gaacgcactg cacttggaca cagccacgcc gcttcccact gactcatttg ggactggcgc 120 cgttgcctgt catgactgtt cgcatcgtcg tcatcaacca tcgactgaca cgcttcgctt 180 tgatttgatt gcttctctct ccactctctc tcttcctgtc tctctactac tactactact 240 ctctcttctg catctccacc ggcctgtgac cgaaaaaacc aactccgtct cctttcgaag 300 aagaaacagt tggtccgacg tcacaagcac attcacaaaa atcaaacaac atatccccat 360 ctttcatata caccacacgc ttatgcagtg agagagcacg agagaagcat cgtcataatc 420 aacacatcag tcaaagcgaa ctgcgctcgg caacacgaca cggcaggcaa catggcgttc 480 cagcagtcgt ctcccctcgt caagtttgag gcctctcccg ccgaatcctt cctctccgcc 540 cccggcgaca acttcacatc cctcttcgcc gactcaacac cctcaacact taaccctcgg 600 gacatgatga cccctgacag cgtcgccgac atcgactctc gcctgtccgt catccccgaa 660 tcacaggacg cggaagatga cgaatcacac tccacatccg ctaccgcacc ctctacctca 720 gaaaagaagc ccgtcaagaa gaggaaatca tggggccagg ttcttcctga gcccaagacc 780 aacctccctc ctcggtatgt cactgcaaca cggctcactt gatacaactt gcatcctaac 840 caaacgttac tgtagaaaac gtgcaaagac ggaagatgaa aaggagcagc gccgcgtcga 900 gcgtgttctc cgcaaccgcc gcgccgcgca gtcctcgcgc gagcgcaaga ggctcgaggt 960 cgaggctctc gagaagcgca acaaggagct cgagacgctc ctcatcaacg tccagaagac 1020 caacctgatc ctcgtcgagg actcaaccgc ttccgacgca gctcaggcgt cgtcacccgc 1080 tcgtcctccc ccctcgactc tctccaggac agcatcactc tctcccagca actctttggc 1140 tcgcgggatg gccaaaccat gtccaacccc gagcagtcct tgatggacca gatcatgaga 1200 tctgccgcta accctaccgt taacccggcc tctctttccc cctccctccc ccccatctcg 1260 gacaaggagt tccagaccaa ggaggaggac gaggaacagg ccgacgaaga tgaagagatg 1320 gagcagacat ggcacgagac caaagaagcc gccgccgcca aggagaagaa cagcaagcag 1380 tcccgcgtct ccactgattc gacacaacgt cctgcagaga tgttgtgcga cccgcagtgt 1440 caatcggtgg agatgccgct gtccctgtct tctcagacga cgccggcgca aactgccttg 1500 gcctggaccc tgttcatcag gatgatggtc ctttcagcat cggccattct ttcggcctgt 1560 cagcggccct tgatgcagat cgctatctcc tcgaaagcca acttctcgct tcgcccaacg 1620 cctcaactgt tgacgacgat tatctggctg gtgactctgc cgcctgcttc acgaatcctc 1680 tcccctccga ctacgacttc gacatcaacg acttcctcac agacgacgca aaccacgccg 1740 cctatgacat tgtggcagcg agcaactatg ccgctgcgga ccgcgagctc gacctcgaga 1800 tccacgaccc tgagaatcag atcccttcgc gacattctat ccagcagccc cagtctggcg 1860 cgtcctctca tggatgcgac gatggcggca ttgcggttgg tgtctgaggg acgcgacgat 1920 cggggcggga tcccggcctc cgagtcttgt gcgacgcgcg gcgactgcga gctggaacgg 1980 tgcctacgca gcgtgacctt gccgtctcga gaagtcctca tcaccctgtg gtgggccgtg 2040 aaggtggagg agaggaggat tcgcctgagg cagcacaaga agcaggccgc ggctctcgac 2100 cccgagaagc gcgcctcctt ggcagacaag aagaaccgac aacaacaaca acaacaacac 2160 cagtatcaga ttccttcgtt ttcaaaatag ttagcatatg tggtttttta atgggcaatg 2220 gggcggatgg caacacggta gaggcaacaa gggttgacta cacctcccaa agggatacgg 2280 cgcacagcga ggttaatgac aaggctaaga tgggcctttt ttttttatga tatgagaacc 2340 tcttcatctc cctttacact tctctctaga tggtagtgat gatatactgt accaaaatac 2400 aacgtctacc tagtgct 2417 2 451 PRT Trichoderma reesei 2 Met Ala Phe Gln Gln Ser Ser Pro Leu Val Lys Phe Glu Ala Ser Pro 1 5 10 15 Ala Glu Ser Phe Leu Ser Ala Pro Gly Asp Asn Phe Thr Ser Leu Phe 20 25 30 Ala Asp Ser Thr Pro Ser Thr Leu Asn Pro Arg Asp Met Met Thr Pro 35 40 45 Asp Ser Val Ala Asp Ile Asp Ser Arg Leu Ser Val Ile Pro Glu Ser 50 55 60 Gln Asp Ala Glu Asp Asp Glu Ser His Ser Thr Ser Ala Thr Ala Pro 65 70 75 80 Ser Thr Ser Glu Lys Lys Pro Val Lys Lys Arg Lys Ser Trp Gly Gln 85 90 95 Val Leu Pro Glu Pro Lys Thr Asn Leu Pro Pro Arg Lys Arg Ala Lys 100 105 110 Thr Glu Asp Glu Lys Glu Gln Arg Arg Val Glu Arg Val Leu Arg Asn 115 120 125 Arg Arg Ala Ala Gln Ser Ser Arg Glu Arg Lys Arg Leu Glu Val Glu 130 135 140 Ala Leu Glu Lys Arg Asn Lys Glu Leu Glu Thr Leu Leu Ile Asn Val 145 150 155 160 Gln Lys Thr Asn Leu Ile Leu Val Glu Glu Leu Asn Arg Phe Arg Arg 165 170 175 Ser Ser Gly Val Val Thr Arg Ser Ser Ser Pro Leu Asp Ser Leu Gln 180 185 190 Asp Ser Ile Thr Leu Ser Gln Gln Leu Phe Gly Ser Arg Asp Gly Gln 195 200 205 Thr Met Ser Asn Pro Glu Gln Ser Leu Met Asp Gln Ile Met Arg Ser 210 215 220 Ala Ala Asn Pro Thr Val Asn Pro Ala Ser Leu Ser Pro Ser Leu Pro 225 230 235 240 Pro Ile Ser Asp Lys Glu Phe Gln Thr Lys Glu Glu Asp Glu Glu Gln 245 250 255 Ala Asp Glu Asp Glu Glu Met Glu Gln Thr Trp His Glu Thr Lys Glu 260 265 270 Ala Ala Ala Ala Lys Glu Lys Asn Ser Lys Gln Ser Arg Val Ser Thr 275 280 285 Asp Ser Thr Gln Arg Pro Ala Val Ser Ile Gly Gly Asp Ala Ala Val 290 295 300 Pro Val Phe Ser Asp Asp Ala Gly Ala Asn Cys Leu Gly Leu Asp Pro 305 310 315 320 Val His Gln Asp Asp Gly Pro Phe Ser Ile Gly His Ser Phe Gly Leu 325 330 335 Ser Ala Ala Leu Asp Ala Asp Arg Tyr Leu Leu Glu Ser Gln Leu Leu 340 345 350 Ala Ser Pro Asn Ala Ser Thr Val Asp Asp Asp Tyr Leu Ala Gly Asp 355 360 365 Ser Ala Ala Cys Phe Thr Asn Pro Leu Pro Ser Asp Tyr Asp Phe Asp 370 375 380 Ile Asn Asp Phe Leu Thr Asp Asp Ala Asn His Ala Ala Tyr Asp Ile 385 390 395 400 Val Ala Ala Ser Asn Tyr Ala Ala Ala Asp Arg Glu Leu Asp Leu Glu 405 410 415 Ile His Asp Pro Glu Asn Gln Ile Pro Ser Arg His Ser Ile Gln Gln 420 425 430 Pro Gln Ser Gly Ala Ser Ser His Gly Cys Asp Asp Gly Gly Ile Ala 435 440 445 Val Gly Val 450 3 1615 DNA Aspergillus nidulans 3 gccatccttg gtgactgagc cccaacactt tcactggtcg ggatagtagc ctctggcttc 60 gattcgctat gacaccgtgg cctctgtcct aagtgactca ggcaaggcaa tcccagttcc 120 aactcccaac ttcgcaacct catcaaccac ctgcttccgt ctagttgcag ttatcagact 180 tgagttgtat gaaatcagca gaccggtttt cgccagtgaa aatggaggac gctttcgcaa 240 actctttgcc tactaccccg tcattggagg ttcctgtgct cactgtctcc ccggctgaca 300 catctcttcg gacgaagaat gtggtggctc agacaaagcc tgaggagaag aagccagcga 360 agaaaagaaa gtcctggggc caggaattac cagttcccaa gacaaactta cctccaaggt 420 gtgtgatacc tcaagagtca actccttact cctgctaata actaccacag aaaacgcgct 480 aagacagaag atgagaaaga gcagcgccgg attgagcgag ttcttcgcaa ccgcgcagcc 540 gcacaaacct ctcgcgagcg caagagactt gaaatggaga agttagaaag cgagaagatt 600 gatatggaac aacaaaacca gttccttctt cagcgtctcg cccagatgga ggctgagaac 660 aaccgtttaa gtcagcaagt tgctcagcta tccgcggagg ttcggggatc ccgccacagc 720 actccaactt ccagttcccc cgcgtcagtt tcgccaactc tcacaccgac tctttttaag 780 caggaagggg atgaggttcc tctggaccgc atcccttttc caactccctc cgtgaccgac 840 tactccccaa ctcttaagcc ttcatctctg gctgagtccc ccgatttgac acaacatcct 900 gcagcgatgt tgtgcgacct gcagtgtcag tcggcgggct cgaaggagat gaaagtgccc 960 tcacgctttt cgacctcgga gccagcatta agcatgagcc tacacatgac cttacagctc 1020 ctctttctga cgatgacttc cgccgcctat tcaacggtga ttcatccctt gagtcagatt 1080 cttcactcct tgaagacggg ttcgcctttg acgttctcga ctcaggagat ttatcagcat 1140 ttccatttga ttctatggtt gattttgaca ccgagcctgt caccctcgaa gatctcgagc 1200 aaaccaacgg cctttcggat tcagcttctt gcaaggctgc tagcttgcaa cccagccatg 1260 gcgcgtccac ttcgcgatgc gacgggcagg gcattgcagc tggcagtgcg tgagaggttt 1320 tcgacggaag accgtctggt tcccgatgtt gtagagggtc gatggagctg ggaatccttg 1380 ttaacgctag cgtcggcgat aaatcttctt gagaaaccgg agcgacgaag aagaaccttg 1440 aggggtcttg attcgttaaa gcggggtcgg cgtattgatt cggggaagcg gtacagggtc 1500 atacggagtt cacggagttc aactagccca agagaggcgt tgacgtctcg gagaaagggc 1560 ttatgataat ttgtatatta gcgtgtccac tattcaatgt aagagcgagc aattg 1615 4 349 PRT Aspergillus nidulans 4 Met Lys Ser Ala Asp Arg Phe Ser Pro Val Lys Met Glu Asp Ala Phe 1 5 10 15 Ala Asn Ser Pro Thr Thr Pro Ser Leu Glu Val Pro Val Leu Thr Val 20 25 30 Ser Pro Ala Asp Thr Ser Leu Arg Thr Lys Asn Val Val Ala Gln Thr 35 40 45 Lys Pro Glu Glu Lys Lys Pro Ala Lys Lys Arg Lys Ser Trp Gly Gln 50 55 60 Glu Leu Pro Val Pro Lys Thr Asn Leu Pro Pro Arg Lys Arg Ala Lys 65 70 75 80 Thr Glu Asp Glu Lys Glu Gln Arg Arg Ile Glu Arg Val Leu Arg Asn 85 90 95 Arg Ala Ala Ala Gln Thr Ser Arg Glu Arg Lys Arg Leu Glu Met Glu 100 105 110 Lys Leu Glu Ser Glu Lys Ile Asp Met Glu Gln Gln Asn Gln Phe Leu 115 120 125 Leu Gln Arg Leu Ala Gln Met Glu Ala Glu Asn Asn Arg Leu Ser Gln 130 135 140 Gln Val Ala Gln Leu Ser Ala Glu Val Arg Gly Ser Arg His Ser Thr 145 150 155 160 Pro Thr Ser Ser Ser Pro Ala Ser Val Ser Pro Thr Leu Thr Pro Thr 165 170 175 Leu Phe Lys Gln Glu Gly Asp Glu Val Pro Leu Asp Arg Ile Pro Phe 180 185 190 Pro Thr Pro Ser Val Thr Asp Tyr Ser Pro Thr Leu Lys Pro Ser Ser 195 200 205 Leu Ala Glu Ser Pro Asp Leu Thr Gln His Pro Ala Val Ser Val Gly 210 215 220 Gly Leu Glu Gly Asp Glu Ser Ala Leu Thr Leu Phe Asp Leu Gly Ala 225 230 235 240 Ser Ile Lys His Glu Pro Thr His Asp Leu Thr Ala Pro Leu Ser Asp 245 250 255 Asp Asp Phe Arg Arg Leu Phe Asn Gly Asp Ser Ser Leu Glu Ser Asp 260 265 270 Ser Ser Leu Leu Glu Asp Gly Phe Ala Phe Asp Val Leu Asp Ser Gly 275 280 285 Asp Leu Ser Ala Phe Pro Phe Asp Ser Met Val Asp Phe Asp Thr Glu 290 295 300 Pro Val Thr Leu Glu Asp Leu Glu Gln Thr Asn Gly Leu Ser Asp Ser 305 310 315 320 Ala Ser Cys Lys Ala Ala Ser Leu Gln Pro Ser His Gly Ala Ser Thr 325 330 335 Ser Arg Cys Asp Gly Gln Gly Ile Ala Ala Gly Ser Ala 340 345 5 451 PRT Trichoderma reesei 5 Met Ala Phe Gln Gln Ser Ser Pro Leu Val Lys Phe Glu Ala Ser Pro 1 5 10 15 Ala Glu Ser Phe Leu Ser Ala Pro Gly Asp Asn Phe Thr Ser Leu Phe 20 25 30 Ala Asp Ser Thr Pro Ser Thr Leu Asn Pro Arg Asp Met Met Thr Pro 35 40 45 Asp Ser Val Ala Asp Ile Asp Ser Arg Leu Ser Val Ile Pro Glu Ser 50 55 60 Gln Asp Ala Glu Asp Asp Glu Ser His Ser Thr Ser Ala Thr Ala Pro 65 70 75 80 Ser Thr Ser Glu Lys Lys Pro Val Lys Lys Arg Lys Ser Trp Gly Gln 85 90 95 Val Leu Pro Glu Pro Lys Thr Asn Leu Pro Pro Arg Lys Arg Ala Lys 100 105 110 Thr Glu Asp Glu Lys Glu Gln Arg Arg Val Glu Arg Val Leu Arg Asn 115 120 125 Arg Arg Ala Ala Gln Ser Ser Arg Glu Arg Lys Arg Leu Glu Val Glu 130 135 140 Ala Leu Glu Lys Arg Asn Lys Glu Leu Glu Thr Leu Leu Ile Asn Val 145 150 155 160 Gln Lys Thr Asn Leu Ile Leu Val Glu Glu Leu Asn Arg Phe Arg Arg 165 170 175 Ser Ser Gly Val Val Thr Arg Ser Ser Ser Pro Leu Asp Ser Leu Gln 180 185 190 Asp Ser Ile Thr Leu Ser Gln Gln Leu Phe Gly Ser Arg Asp Gly Gln 195 200 205 Thr Met Ser Asn Pro Glu Gln Ser Leu Met Asp Gln Ile Met Arg Ser 210 215 220 Ala Ala Asn Pro Thr Val Asn Pro Ala Ser Leu Ser Pro Ser Leu Pro 225 230 235 240 Pro Ile Ser Asp Lys Glu Phe Gln Thr Lys Glu Glu Asp Glu Glu Gln 245 250 255 Ala Asp Glu Asp Glu Glu Met Glu Gln Thr Trp His Glu Thr Lys Glu 260 265 270 Ala Ala Ala Ala Lys Glu Lys Asn Ser Lys Gln Ser Arg Val Ser Thr 275 280 285 Asp Ser Thr Gln Arg Pro Ala Val Ser Ile Gly Gly Asp Ala Ala Val 290 295 300 Pro Val Phe Ser Asp Asp Ala Gly Ala Asn Cys Leu Gly Leu Asp Pro 305 310 315 320 Val His Gln Asp Asp Gly Pro Phe Ser Ile Gly His Ser Phe Gly Leu 325 330 335 Ser Ala Ala Leu Asp Ala Asp Arg Tyr Leu Leu Glu Ser Gln Leu Leu 340 345 350 Ala Ser Pro Asn Ala Ser Thr Val Asp Asp Asp Tyr Leu Ala Gly Asp 355 360 365 Ser Ala Ala Cys Phe Thr Asn Pro Leu Pro Ser Asp Tyr Asp Phe Asp 370 375 380 Ile Asn Asp Phe Leu Thr Asp Asp Ala Asn His Ala Ala Tyr Asp Ile 385 390 395 400 Val Ala Ala Ser Asn Tyr Ala Ala Ala Asp Arg Glu Leu Asp Leu Glu 405 410 415 Ile His Asp Pro Glu Asn Gln Ile Pro Ser Arg His Ser Ile Gln Gln 420 425 430 Pro Gln Ser Gly Ala Ser Ser His Gly Cys Asp Asp Gly Gly Ile Ala 435 440 445 Val Gly Val 450 6 349 PRT Aspergillus nidulans 6 Met Lys Ser Ala Asp Arg Phe Ser Pro Val Lys Met Glu Asp Ala Phe 1 5 10 15 Ala Asn Ser Pro Thr Thr Pro Ser Leu Glu Val Pro Val Leu Thr Val 20 25 30 Ser Pro Ala Asp Thr Ser Leu Arg Thr Lys Asn Val Val Ala Gln Thr 35 40 45 Lys Pro Glu Glu Lys Lys Pro Ala Lys Lys Arg Lys Ser Trp Gly Gln 50 55 60 Glu Leu Pro Val Pro Lys Thr Asn Leu Pro Pro Arg Lys Arg Ala Lys 65 70 75 80 Thr Glu Asp Glu Lys Glu Gln Arg Arg Ile Glu Arg Val Leu Arg Asn 85 90 95 Arg Ala Ala Ala Gln Thr Ser Arg Glu Arg Lys Arg Leu Glu Met Glu 100 105 110 Lys Leu Glu Ser Glu Lys Ile Asp Met Glu Gln Gln Asn Gln Phe Leu 115 120 125 Leu Gln Arg Leu Ala Gln Met Glu Ala Glu Asn Asn Arg Leu Ser Gln 130 135 140 Gln Val Ala Gln Leu Ser Ala Glu Val Arg Gly Ser Arg His Ser Thr 145 150 155 160 Pro Thr Ser Ser Ser Pro Ala Ser Val Ser Pro Thr Leu Thr Pro Thr 165 170 175 Leu Phe Lys Gln Glu Gly Asp Glu Val Pro Leu Asp Arg Ile Pro Phe 180 185 190 Pro Thr Pro Ser Val Thr Asp Tyr Ser Pro Thr Leu Lys Pro Ser Ser 195 200 205 Leu Ala Glu Ser Pro Asp Leu Thr Gln His Pro Ala Val Ser Val Gly 210 215 220 Gly Leu Glu Gly Asp Glu Ser Ala Leu Thr Leu Phe Asp Leu Gly Ala 225 230 235 240 Ser Ile Lys His Glu Pro Thr His Asp Leu Thr Ala Pro Leu Ser Asp 245 250 255 Asp Asp Phe Arg Arg Leu Phe Asn Gly Asp Ser Ser Leu Glu Ser Asp 260 265 270 Ser Ser Leu Leu Glu Asp Gly Phe Ala Phe Asp Val Leu Asp Ser Gly 275 280 285 Asp Leu Ser Ala Phe Pro Phe Asp Ser Met Val Asp Phe Asp Thr Glu 290 295 300 Pro Val Thr Leu Glu Asp Leu Glu Gln Thr Asn Gly Leu Ser Asp Ser 305 310 315 320 Ala Ser Cys Lys Ala Ala Ser Leu Gln Pro Ser His Gly Ala Ser Thr 325 330 335 Ser Arg Cys Asp Gly Gln Gly Ile Ala Ala Gly Ser Ala 340 345 7 1265 DNA Aspergillus nidulans 7 tttgaacagc agatcgttac tgcctaccca gacgttacag tccacgagct cacggaggac 60 gatgaattct tagtaatcgc ttgcgatggt gggtttcccc tcaactttgc cgctctgttc 120 cacaatctga tatactacag gaatctggga ttgccagtct tcccaagccg tggtcgaatt 180 cgttcgccgc ggtatcgcgg ccaagcagga tctctatcgg atttgtgaaa acatgatgga 240 caactgtctc gcttccaaca gtgagactgg tggagttggc tgtgacaaca tgacaatggt 300 cattataggt ctcctcaatg gaaaaactaa ggaagagtgg tacaaccaga tcgcggagcg 360 ggttgctaac ggcgacggcc cttgtgctcc gcccgaatac ggcaagtctc tcgaggaacc 420 cacggcctcc aatccctact gactgaaccg tgggggttgc agctgaattc cgaggacctg 480 gaatccataa ccattttgaa gagaacccgg acgagtacga gatcgaccac gatcgctccc 540 gcccattcaa cgtgcgttct ggtagaataa ttcttttggg agatggcagc acgttaattc 600 caggaaaaca gaatgacgag gaactctttg accaaaccgg ggaggagaat cacccagacc 660 aagtgcaacg ccagaatacc gacacagaaa gaaatgaccg tgaagggacg cctgggcctc 720 aatccgcggc tccccagacg aacacgtccg cttcggatgg ctcagagcct tctaacacac 780 cgcagaaacc cgcctcttcg tagcttcgtc atgagattta cgcctgattc ccttcatttt 840 ggttcctgaa acgactcgtg atttcacgat ccacacccgc cgccccatct ccacgcccgg 900 tgccgaagcc tcacaattct gcccccatac ggtcgctcat tgattttctg tttctcacga 960 tttgaaggcg cattggtgct tgtgaccgcg aagatgcgaa agagacggac catatcatcc 1020 ccttctatct cttgttttaa tcccatcttc ttacttttta cgagctcatc cagatcaaat 1080 caccttcgtg ttactccagg atggatatct ttgagaattc gccgaatggg tggaggcatc 1140 ttctttccct gtcatctttc ttctctatgt ttgcacatgc cgcaagcggc aggcctcacg 1200 agagtacgtt tgtttcatgt ctcgacataa gataccgcaa caaccactat tgacgaactt 1260 tataa 1265 8 130 PRT Aspergillus nidulans 8 Phe Glu Gln Gln Ile Val Thr Ala Tyr Pro Asp Val Thr Val His Glu 1 5 10 15 Leu Thr Glu Asp Asp Glu Phe Leu Val Ile Ala Cys Asp Gly Gly Ile 20 25 30 Trp Asp Cys Gln Ser Ser Gln Ala Val Val Glu Phe Val Arg Arg Gly 35 40 45 Ile Ala Ala Lys Gln Asp Leu Tyr Arg Ile Cys Glu Asn Met Met Asp 50 55 60 Asn Cys Leu Ala Ser Asn Ser Glu Thr Gly Gly Val Gly Cys Asp Asn 65 70 75 80 Met Thr Met Val Ile Ile Gly Leu Leu Asn Gly Lys Thr Lys Glu Glu 85 90 95 Trp Tyr Asn Gln Ile Ala Glu Arg Val Ala Asn Gly Asp Gly Pro Cys 100 105 110 Ala Pro Pro Glu Tyr Gly Lys Ser Leu Glu Glu Pro Thr Ala Ser Asn 115 120 125 Pro Tyr 130 9 1824 DNA Trichoderma reesei 9 gacgagcctc gatccgcctc gacgccgctg gtttccccct tctttctccc cccttcagcc 60 acgtcctcgt gtcctataac ctttcgcagc ctacggtccc gcctccagag gtctcgcgtc 120 cctgagtacc aaacgataga aacaagactg ctatctttgt cgtgctgcct cctcccctcc 180 tcgacgcttt tcctccccct cgatcgcttt cccggccctc gtgagacgtc gcagccatgg 240 gccaaaccct ctcggagccc gttgtcgaaa agacttccga aaagggcgag gatgacagac 300 tcatctacgg cgtgtccgcc atgcagggct ggcgcatcag catggaggac gctcacacgg 360 ctgagctgaa tctcccccca cctgacaacg acaccaagac gcaccccgac aggctgtcct 420 ttttcggagt cttcgacgga cacggaggag acaaagtagc gttattcgca ggcgagaaca 480 ttcacaacat tgttttcaag caggagagct tcaaatccgg tgattacgct cagggtctca 540 aggacggctt tctcgctacg gatcgggcta ttctcaacga ccccaaatac gaagaggaag 600 tctctggctg cactgcctgc gtcaccctga ttgccggaaa caaactatat gtcgccaacg 660 ccggtgattc tcgaagcgtg ctgggcatca agggacgggc caaaccccta tccaacgacc 720 acaagcctca gcttgaaacg gagaagaacc gaatcacagc cgctggcggt ttcgtcgact 780 ttggccgagt caacggcaat ctggctctgt cgcgtgccat tggcgacttt gaattcaaga 840 agagcgccga gctgtccccc gaaaaccaga tcgttaccgc ctttcccgat gtcgaggtgc 900 acgagcttac agaggaggac gagttcctgg tgattgcctg tgacggtatc tgggattgcc 960 aatcttccca ggctgttgtt gagtttgtgc gacgaggcat cgccgccaag caggaccttg 1020 acaagatctg cgagaacatg atggacaact gccttgcgtc caactcagaa acgggtggcg 1080 tcggctgcga caacatgacc atggtcatca tcggcttcct gcacggcaag accaaggagg 1140 agtggtatga cgaaattgcc aagagagtgg ccaacggaga cggcccctgt gcccccccgg 1200 aatatgccga gttccgcggt cccggcgttc accacaacta cgaagacagc gacagcggct 1260 acgacgtcga cgccgacagc ggcggcaagt ttagccttgc cggatcccgg ggtcgcatca 1320 tcttcctggg cgacggcacc gaagtcctga cgggctccga cgacacggag atgtttgaca 1380 atgctgacga ggacaaggac cttgcgagcc aggtgcccaa gagctccggc aagaccgatg 1440 caaaggagga gacagaggcc aagccggcac cagaggcgga gtcgtccaaa cccgcggatg 1500 ggtcggagaa gaagcaagac gaaaagacac ccgaggagag taagaaggat taggtggtcc 1560 tcttgaattc tttgggctcg tctccttgaa gccccgcgct ggtgttgttg atggcgtgtg 1620 tttgtgtgta cgtgtggcat aattcttttt tcttcccatc accgctactc aaaaaacccc 1680 aggcgtgagg gcatttttaa atcgcatagg gagtggggga gagacgggag aggctctgga 1740 acgaaacatt ctgggagaca aggcagagag cgtaggggcg gtttagacat tgagtgttgc 1800 tcgttaaaaa aaaaaaaaaa aaaa 1824 10 438 PRT Trichoderma reesei 10 Met Gly Gln Thr Leu Ser Glu Pro Val Val Glu Lys Thr Ser Glu Lys 1 5 10 15 Gly Glu Asp Asp Arg Leu Ile Tyr Gly Val Ser Ala Met Gln Gly Trp 20 25 30 Arg Ile Ser Met Glu Asp Ala His Thr Ala Glu Leu Asn Leu Pro Pro 35 40 45 Pro Asp Asn Asp Thr Lys Thr His Pro Asp Arg Leu Ser Phe Phe Gly 50 55 60 Val Phe Asp Gly His Gly Gly Asp Lys Val Ala Leu Phe Ala Gly Glu 65 70 75 80 Asn Ile His Asn Ile Val Phe Lys Gln Glu Ser Phe Lys Ser Gly Asp 85 90 95 Tyr Ala Gln Gly Leu Lys Asp Gly Phe Leu Ala Thr Asp Arg Ala Ile 100 105 110 Leu Asn Asp Pro Lys Tyr Glu Glu Glu Val Ser Gly Cys Thr Ala Cys 115 120 125 Val Thr Leu Ile Ala Gly Asn Lys Leu Tyr Val Ala Asn Ala Gly Asp 130 135 140 Ser Arg Ser Val Leu Gly Ile Lys Gly Arg Ala Lys Pro Leu Ser Asn 145 150 155 160 Asp His Lys Pro Gln Leu Glu Thr Glu Lys Asn Arg Ile Thr Ala Ala 165 170 175 Gly Gly Phe Val Asp Phe Gly Arg Val Asn Gly Asn Leu Ala Leu Ser 180 185 190 Arg Ala Ile Gly Asp Phe Glu Phe Lys Lys Ser Ala Glu Leu Ser Pro 195 200 205 Glu Asn Gln Ile Val Thr Ala Phe Pro Asp Val Glu Val His Glu Leu 210 215 220 Thr Glu Glu Asp Glu Phe Leu Val Ile Ala Cys Asp Gly Ile Trp Asp 225 230 235 240 Cys Gln Ser Ser Gln Ala Val Val Glu Phe Val Arg Arg Gly Ile Ala 245 250 255 Ala Lys Gln Asp Leu Asp Lys Ile Cys Glu Asn Met Met Asp Asn Cys 260 265 270 Leu Ala Ser Asn Ser Glu Thr Gly Gly Val Gly Cys Asp Asn Met Thr 275 280 285 Met Val Ile Ile Gly Phe Leu His Gly Lys Thr Lys Glu Glu Trp Tyr 290 295 300 Asp Glu Ile Ala Lys Arg Val Ala Asn Gly Asp Gly Pro Cys Ala Pro 305 310 315 320 Pro Glu Tyr Ala Glu Phe Arg Gly Pro Gly Val His His Asn Tyr Glu 325 330 335 Asp Ser Asp Ser Gly Tyr Asp Val Asp Ala Asp Ser Gly Gly Lys Phe 340 345 350 Ser Leu Ala Gly Ser Arg Gly Arg Ile Ile Phe Leu Gly Asp Gly Thr 355 360 365 Glu Val Leu Thr Gly Ser Asp Asp Thr Glu Met Phe Asp Asn Ala Asp 370 375 380 Glu Asp Lys Asp Leu Ala Ser Gln Val Pro Lys Ser Ser Gly Lys Thr 385 390 395 400 Asp Ala Lys Glu Glu Thr Glu Ala Lys Pro Ala Pro Glu Ala Glu Ser 405 410 415 Ser Lys Pro Ala Asp Gly Ser Glu Lys Lys Gln Asp Glu Lys Thr Pro 420 425 430 Glu Glu Ser Lys Lys Asp 435 11 1570 DNA Aspergillus nidulans 11 cggaggcaag agtcatagac gcgggaagaa gaaaattgag agtgagaaag aggaatctga 60 tcacgcccct ggcaccttgc aacccccggc tgggcccgat gccgggttag ctctcacccg 120 cactgcatct aatgaggtgt ttgaagcgga cggtgtcatc cagattggcc gtttgaaggt 180 ctttacggct gacgttctgg gtcatggaag ccacgggaca gttgtttacc gcgggtcgtt 240 tgacggccga gacgtcgcgg tcaaacgtat gctggtggag ttctatgata ttgcatcgca 300 cgaagtggga ttgttgcagg aaagcgatga tcataacaac gttatccgat gttattgccg 360 tgagcaagcc aagggtttct tctacatcgc ccttgaactg tgtccggctt ctttgcagga 420 tgtggtagaa cgaccagacg cgttcccgca gctagtcaat ggtggcttgg atatgccgga 480 cgtcttgcgt caaattgtcg ccggtgtccg gtacctacac tctctcaaaa tcgtacaccg 540 tgacttgaag cctcaaaata tcctggtcgc cgctcctcga ggccgtatcg gttctcgggc 600 catccggctt ctgatttcgg actttggctt gtgcaagaaa cttgaggata accagagttc 660 attcagggca accacggccc atgctgctgg tactccgggt ggagggctcc cgaactgctt 720 gtggatgacg acaagagccg gtaatcaggg ttcagagtct caaaatacgg agtcatctga 780 gccggcggtc gtcgatcccc agacgaatcg acgagccacc cgagccattg atatcttctc 840 cctgggatgt gtcttctact acgtcctaac tcgaggatgt catccttttg acaagaatgg 900 caagttcatg cgcgaagcaa atatcgtcaa ggggaatttc aatctcgatg agttacagcg 960 tctaggagag tatgcgtttg aagcagacga tcttatccga tcaatgttgg cacttgatcc 020 acgtcaacgg tatgtcccaa caacatcttc ctttgccttg tggcgtagcg tactaatctc 080 cacagccccg acgcaagcgc tgtgttaacc catcctttct tctggaatcc gtccgaccgc 140 cttagcttcc tctgtgacgt ttcggaccac ttcgagttcg aaccgagaga tcctccatct 200 gacgctcttc tgtgtctaga gtctgtagcc tctgatgtca ttggccctga aatgaatcct 260 caaactcctg ccaaaggact tcaaagacag tctcggaagc agcgaaaata caccggctcc 320 aaaatgctgg acttgatgcg agccctgcgg aacaagcgca accactacaa tgatatgccg 380 gagcatttga aagctcatat tggtgggctg ccggagggtt acttgaattt ctggaccgtg 440 cgtttcccga gtttgctgat gagttgtcat tgggtgattg ttgaactggg attgacgaag 500 acggatcggt tccaagagat attttacgcc attggagtag gttgttgcgt actggttcag 560 aaatatattg 570 12 504 PRT Aspergillus nidulans 12 Gly Gly Lys Ser His Arg Arg Gly Lys Lys Lys Ile Glu Ser Glu Lys 1 5 10 15 Glu Glu Ser Asp His Ala Pro Gly Thr Leu Gln Pro Pro Ala Gly Pro 20 25 30 Asp Ala Gly Leu Ala Leu Thr Arg Thr Ala Ser Asn Glu Val Phe Glu 35 40 45 Ala Asp Gly Val Ile Gln Ile Gly Arg Leu Lys Val Phe Thr Ala Asp 50 55 60 Val Leu Gly His Gly Ser His Gly Thr Val Val Tyr Arg Gly Ser Phe 65 70 75 80 Asp Gly Arg Asp Val Ala Val Lys Arg Met Leu Val Glu Phe Tyr Asp 85 90 95 Ile Ala Ser His Glu Val Gly Leu Leu Gln Glu Ser Asp Asp His Asn 100 105 110 Asn Val Ile Arg Cys Tyr Cys Arg Glu Gln Ala Lys Gly Phe Phe Tyr 115 120 125 Ile Ala Leu Glu Leu Cys Pro Ala Ser Leu Gln Asp Val Val Glu Arg 130 135 140 Pro Asp Ala Phe Pro Gln Leu Val Asn Gly Gly Leu Asp Met Pro Asp 145 150 155 160 Val Leu Arg Gln Ile Val Ala Gly Val Arg Tyr Leu His Ser Leu Lys 165 170 175 Ile Val His Arg Asp Leu Lys Pro Gln Asn Ile Leu Val Ala Ala Pro 180 185 190 Arg Gly Arg Ile Gly Ser Arg Ala Ile Arg Leu Leu Ile Ser Asp Phe 195 200 205 Gly Leu Cys Lys Lys Leu Glu Asp Asn Gln Ser Ser Phe Arg Ala Thr 210 215 220 Thr Ala His Ala Ala Gly Thr Pro Gly Gly Gly Leu Pro Asn Cys Leu 225 230 235 240 Trp Met Thr Thr Arg Ala Gly Asn Gln Gly Ser Glu Ser Gln Asn Thr 245 250 255 Glu Ser Ser Glu Pro Ala Val Val Asp Pro Gln Thr Asn Arg Arg Ala 260 265 270 Thr Arg Ala Ile Asp Ile Phe Ser Leu Gly Cys Val Phe Tyr Tyr Val 275 280 285 Leu Thr Arg Gly Cys His Pro Phe Asp Lys Asn Gly Lys Phe Met Arg 290 295 300 Glu Ala Asn Ile Val Lys Gly Asn Phe Asn Leu Asp Glu Leu Gln Arg 305 310 315 320 Leu Gly Glu Tyr Ala Phe Glu Ala Asp Asp Leu Ile Arg Ser Met Leu 325 330 335 Ala Leu Asp Pro Arg Gln Arg Pro Asp Ala Ser Ala Val Leu Thr His 340 345 350 Pro Phe Phe Trp Asn Pro Ser Asp Arg Leu Ser Phe Leu Cys Asp Val 355 360 365 Ser Asp His Phe Glu Phe Glu Pro Arg Asp Pro Pro Ser Asp Ala Leu 370 375 380 Leu Cys Leu Glu Ser Val Ala Ser Asp Val Ile Gly Pro Glu Met Asn 385 390 395 400 Pro Gln Thr Pro Ala Lys Gly Leu Gln Arg Gln Ser Arg Lys Gln Arg 405 410 415 Lys Tyr Thr Gly Ser Lys Met Leu Asp Leu Met Arg Ala Leu Arg Asn 420 425 430 Lys Arg Asn His Tyr Asn Asp Met Pro Glu His Leu Lys Ala His Ile 435 440 445 Gly Gly Leu Pro Glu Gly Tyr Leu Asn Phe Trp Thr Val Arg Phe Pro 450 455 460 Ser Leu Leu Met Ser Cys His Trp Val Ile Val Glu Leu Gly Leu Thr 465 470 475 480 Lys Thr Asp Arg Phe Gln Glu Ile Phe Tyr Ala Ile Gly Val Gly Cys 485 490 495 Cys Val Leu Val Gln Lys Tyr Ile 500 13 4528 DNA Trichoderma reesei 13 gcacgagcaa gatacggcct ctcgcaccaa ggagacacgc atattcgtgg taccatcggc 60 tgagggtgaa ggggggttca acacagcaca actcagcgac cactggactg gtggagccga 120 agcccacgat cgaatccaca gcctgcacca ctttctcctc gtcatattcg cggggactca 180 caagcggttt ccgttgcctt cgaattcgac agagctgcga ctgcgagtca tttcagcgac 240 tctaaaccta ctcctttggc tgctgcgcgg gactggttct gcccagcctc tcctactcga 300 ccaaccgacg tcctctttct gcttcctcat ccctttctcc tttgacgtcc gagcgtcaga 360 gcgaattttt ccttgcttct tcgtttgggc cgggaatggc ttctctggca tcgcaacagc 420 ctctacctct ccgttggtag agccatagcc tgcagctccc catgtgatcc gctctccgtc 480 tctccggcac cccgactttc gtctcgatca tgatgcggcg acccccgagc caaggacgat 540 ggtccgcgtc gcatcagaag ctctcctggc ttttgccttt attctcatac catggctcca 600 acttgccgat gctcagcagc agcctcagca gccccagatt cgaattcact cacaaagagg 660 cgacgcgccc cttgacaaag tcgccgacga tgccaacacc cgttggtacg caacacatgc 720 tgcaccagac gtgcaccccg aagcgaagtt cgacaccgtc aacaggaagc aaaagcagca 780 gtcgaccgct tcgccccagc aacaccagaa atatcgacga gccccctatg actacgccag 840 caaggacaag gcccagaacc gatatgcgca gcaccctatc cgcgaatccg agaaaccaaa 900 ctacgtaaaa gtccccaacg atgcgagcgc cctcgcaact ttagctccgg ctcagcccgt 960 ccgagcacca cacacctcac gacatcactg gcccagcagc agcgccgctt ctgggctggc 1020 ctcgccgcac aatgcgcgga gtctggagga ctgggaagtt gaagactttg ttcttctggc 1080 gaccgtcgat ggagacctct atgccagcga ccgaaagacc ggtcggcacc tctggcacct 1140 cgaggtcgac cagccagtgg ttgaaaccaa acactaccga acaaacaact ccgtcctcga 1200 cgacgactat cgccccgtcg accactacat ctgggccgtc gagccgagcc gcgatggagg 1260 gctctatgta tggatccccg actccggagc gggcctcgtc aggaccggct tcaccatgaa 1320 gcacctcgtt gaagaacttg ctccatacgc cggcgacgag ccccccgttg tctataccgg 1380 agacaagaag acgaccatgg tcaccctgga cgccgctacc gggcgcgttc tcaaatggtt 1440 tggctctagc ggctcccaag tcaacgaagc cgagagctgc cttcggccca atgcctttga 1500 cgacagggat accacagagt gcagctccat gggcacaatc acgctgggaa ggaccgagta 1560 cacggtgggc atccagaggc gagacggtcg ccctatcgca accttgaagt acgcagaatg 1620 gggacccaac acctttgaca gcgacctcta ccagcaatac cacgcctcgt tggacaacca 1680 ttacatcacc agtcagcacg acgggagaat ttacgcgttt gacaagtcac aggcagaaaa 1740 cgacctgccc ctctacaccc acaagttttc gtctcccgtc gcccgggtct tcgatgtctg 1800 tcgaccgtgg gatgcgaatg cgggaagcaa cccggagctg gtggttctcc cccaacctcc 1860 aattccagcg cttgatgaga gcactgtcaa gatgcgaagc aacagcatct tcctcaacca 1920 gactgaaagc ggcgactggt atgcgctctc cggccgtgcg tatccgctta tactcgatgc 1980 ccccgtggcc cagatctcgc gggacgactt gtgggatatg gcccatgcct ttgattccat 2040 taacccaaat aagctgtcca aggccctggt gggaacccac tttctgaatc ccgtcaagag 2100 caccggttac catcagccgc cgacgctccc tgccggcgcc ctcgacgagt attacgagga 2160 cttggagaac gcctcaaaca atgctcacgc cgtgacaaac actgttccgg aggagcccac 2220 catcatcacc aaagtcaagg ctcttccgca gagtgctgcg aacagcgtca ttgactttgt 2280 cagcaacccc attctcatca ttttcttgat aggctccttg atctacaacg aaaagaagct 2340 gcgacggtcg tatcatcggt tccggactca tggcacaatc aaggacgtct atcccttctt 2400 cgttatcgaa tctgaggccg gagatgaatc aggtgatgac aaggacggtg tgttcccatc 2460 ttcgccgtct ccgcgcagtc aaccccagga ccaaaatgcg gaagaccacc tgtccagaca 2520 caaggtggag aggaatgccg gcgaccagga caaggtcaag gacaacagga gcctgcatga 2580 cgtttctgac accttggaac cgagcaacaa gactgttgag aaaacggccg atgtggtcaa 2640 gcaagtggat gtagctggcc ctgacgcacc ctcgacggac tccaatggtg ctgcaccgga 2700 gaagaagaag aaggctcacc gaggccgtcg tggcggtgtc aagcacagaa agggtcggcc 2760 caccgacggc tcgcagtctc atgaaaacga cccagctctc actacagtgg acgaggctgt 2820 aagcaatgcg aagaagctgg gtgaccggcc aagcctggaa cccgacgtca tgaccatcta 2880 caacgacatg caagccgtca cgggctctgt tatcagcatg ggaaacatcg aggtcgatac 2940 ggatgtcgag cttggcatgg gcagcaacgg tactgtcgta tttgctggcc gattcgatgg 3000 cagggacgtc gccgtcaaga gaatgacgat tcagttctac gacattgcca cgcgagaaac 3060 taagttgctg cgcgagagtg acgaccaccc caatgtaaat cagccctcat cgtttcaccc 3120 attttccctt cgctaacgta accactgtct gcacgtcatt cggtattact cacaagtgca 3180 gcgaggcgac ttcctgtata ttgccttgga acgctgcgct gcttcattgg cagatgtcat 3240 tgaaaagccg tatgcctttg gtgaattggc caaggctgga caaaaggacc taccgggcgt 3300 cttgtaccaa atcaccaacg gcatcagcca cttgcactct ctgcggattg ttcatcgaga 3360 cttgaagcct caaaacatct tggtcaactt ggacaaggac ggcagaccaa ggctcttggt 3420 gtcggacttt ggcctgtgta agaaactgga ggatagacag tcttcgttcg gagcaacgac 3480 aggccgagcc gctggaacgt cgggatggcg tgcccccgaa ctgcttctcg atgacgacgg 3540 acagaatccc gcagccatcg atagcagtac gcacagcggc tctcacacca tcctcgtggg 3600 agaccccaac tcgctttcca atggagggcg agccacgagg gccattgaca tcttctccct 3660 tggccttgtc ttcttctacg tgctcaccaa tggatcccac ccgtttgact gtggcgacag 3720 atatatgcgg gaggtgaaca ttcgaaaggg caactacaat ctcgatccat tggacgctct 3780 gggcgacttt gcctacgaag ccaaggatct gattgcgtcc atgctccagg cctctcccaa 3840 ggcacgaccc gactcgcgag aggtcatggc ccaccctttc ttctggtctc cgaagaagcg 3900 tctggccttt ttgtgcgacg tgtcggattc tctggagaag gaggtgcgag atcctccgtc 3960 gcctgccttg gtcgagctgg agcgacatgc gccggaggtc attaagggag acttcttgaa 4020 ggtgctcacg cgcgactttg tcgagtcgct gggcaagcag cgcaagtaca ccgggaacaa 4080 gctgctcgac ctgttgcgcg ctcttcgcaa caagcggaat cactacgaag acatgtcgga 4140 ctcgctgaag cgcagcgtgg gatcactgcc tgatgggtat cttgcttatt ggacggtcaa 4200 gttcccgatg ctgttgctga cgtgctggaa cgtggtgtat aatctcgagt gggagaagac 4260 ggatcggttc agggagtact atgagcctgc cggattgtag aagaaagaaa aggaagagaa 4320 aagaaaggcc tcttgcttgt ttggttgctg tatatctttt tgctcgaaga tggaaacgga 4380 aaatattggg gaagttgcat gggaagtgaa caaaagaggg gaaaaatggt gaatgtgaaa 4440 gcaaagtcgg ttagcgggtg ggcatggtcg tcatccatgt aattgtttca gcttctgttg 4500 catcaaaagc gttgtgtttt cgttcttt 4528 14 1232 PRT Trichoderma reesei 14 Met Val Arg Val Ala Ser Glu Ala Leu Leu Ala Phe Ala Phe Ile Leu 1 5 10 15 Ile Pro Trp Leu Gln Leu Ala Asp Ala Gln Gln Gln Pro Gln Gln Pro 20 25 30 Gln Ile Arg Ile His Ser Gln Arg Gly Asp Ala Pro Leu Asp Lys Val 35 40 45 Ala Asp Asp Ala Asn Thr Arg Trp Tyr Ala Thr His Ala Ala Pro Asp 50 55 60 Val His Pro Glu Ala Lys Phe Asp Thr Val Asn Arg Lys Gln Lys Gln 65 70 75 80 Gln Ser Thr Ala Ser Pro Gln Gln His Gln Lys Tyr Arg Arg Ala Pro 85 90 95 Tyr Asp Tyr Ala Ser Lys Asp Lys Ala Gln Asn Arg Tyr Ala Gln His 100 105 110 Pro Ile Arg Glu Ser Glu Lys Pro Asn Tyr Val Lys Val Pro Asn Asp 115 120 125 Ala Ser Ala Leu Ala Thr Leu Ala Pro Ala Gln Pro Val Arg Ala Pro 130 135 140 His Thr Ser Arg His His Trp Pro Ser Ser Ser Ala Ala Ser Gly Leu 145 150 155 160 Ala Ser Pro His Asn Ala Arg Ser Leu Glu Asp Trp Glu Val Glu Asp 165 170 175 Phe Val Leu Leu Ala Thr Val Asp Gly Asp Leu Tyr Ala Ser Asp Arg 180 185 190 Lys Thr Gly Arg His Leu Trp His Leu Glu Val Asp Gln Pro Val Val 195 200 205 Glu Thr Lys His Tyr Arg Thr Asn Asn Ser Val Leu Asp Asp Asp Tyr 210 215 220 Arg Pro Val Asp His Tyr Ile Trp Ala Val Glu Pro Ser Arg Asp Gly 225 230 235 240 Gly Leu Tyr Val Trp Ile Pro Asp Ser Gly Ala Gly Leu Val Arg Thr 245 250 255 Gly Phe Thr Met Lys His Leu Val Glu Glu Leu Ala Pro Tyr Ala Gly 260 265 270 Asp Glu Pro Pro Val Val Tyr Thr Gly Asp Lys Lys Thr Thr Met Val 275 280 285 Thr Leu Asp Ala Ala Thr Gly Arg Val Leu Lys Trp Phe Gly Ser Ser 290 295 300 Gly Ser Gln Val Asn Glu Ala Glu Ser Cys Leu Arg Pro Asn Ala Phe 305 310 315 320 Asp Asp Arg Asp Thr Thr Glu Cys Ser Ser Met Gly Thr Ile Thr Leu 325 330 335 Gly Arg Thr Glu Tyr Thr Val Gly Ile Gln Arg Arg Asp Gly Arg Pro 340 345 350 Ile Ala Thr Leu Lys Tyr Ala Glu Trp Gly Pro Asn Thr Phe Asp Ser 355 360 365 Asp Leu Tyr Gln Gln Tyr His Ala Ser Leu Asp Asn His Tyr Ile Thr 370 375 380 Ser Gln His Asp Gly Arg Ile Tyr Ala Phe Asp Lys Ser Gln Ala Glu 385 390 395 400 Asn Asp Leu Pro Leu Tyr Thr His Lys Phe Ser Ser Pro Val Ala Arg 405 410 415 Val Phe Asp Val Cys Arg Pro Trp Asp Ala Asn Ala Gly Ser Asn Pro 420 425 430 Glu Leu Val Val Leu Pro Gln Pro Pro Ile Pro Ala Leu Asp Glu Ser 435 440 445 Thr Val Lys Met Arg Ser Asn Ser Ile Phe Leu Asn Gln Thr Glu Ser 450 455 460 Gly Asp Trp Tyr Ala Leu Ser Gly Arg Ala Tyr Pro Leu Ile Leu Asp 465 470 475 480 Ala Pro Val Ala Gln Ile Ser Arg Asp Asp Leu Trp Asp Met Ala His 485 490 495 Ala Phe Asp Ser Ile Asn Pro Asn Lys Leu Ser Lys Ala Leu Val Gly 500 505 510 Thr His Phe Leu Asn Pro Val Lys Ser Thr Gly Tyr His Gln Pro Pro 515 520 525 Thr Leu Pro Ala Gly Ala Leu Asp Glu Tyr Tyr Glu Asp Leu Glu Asn 530 535 540 Ala Ser Asn Asn Ala His Ala Val Thr Asn Thr Val Pro Glu Glu Pro 545 550 555 560 Thr Ile Ile Thr Lys Val Lys Ala Leu Pro Gln Ser Ala Ala Asn Ser 565 570 575 Val Ile Asp Phe Val Ser Asn Pro Ile Leu Ile Ile Phe Leu Ile Gly 580 585 590 Ser Leu Ile Tyr Asn Glu Lys Lys Leu Arg Arg Ser Tyr His Arg Phe 595 600 605 Arg Thr His Gly Thr Ile Lys Asp Val Tyr Pro Phe Phe Val Ile Glu 610 615 620 Ser Glu Ala Gly Asp Glu Ser Gly Asp Asp Lys Asp Gly Val Phe Pro 625 630 635 640 Ser Ser Pro Ser Pro Arg Ser Gln Pro Gln Asp Gln Asn Ala Glu Asp 645 650 655 His Leu Ser Arg His Lys Val Glu Arg Asn Ala Gly Asp Gln Asp Lys 660 665 670 Val Lys Asp Asn Arg Ser Leu His Asp Val Ser Asp Thr Leu Glu Pro 675 680 685 Ser Asn Lys Thr Val Glu Lys Thr Ala Asp Val Val Lys Gln Val Asp 690 695 700 Val Ala Gly Pro Asp Ala Pro Ser Thr Asp Ser Asn Gly Ala Ala Pro 705 710 715 720 Glu Lys Lys Lys Lys Ala His Arg Gly Arg Arg Gly Gly Val Lys His 725 730 735 Arg Lys Gly Arg Pro Thr Asp Gly Ser Gln Ser His Glu Asn Asp Pro 740 745 750 Ala Leu Thr Thr Val Asp Glu Ala Val Ser Asn Ala Lys Lys Leu Gly 755 760 765 Asp Arg Pro Ser Leu Glu Pro Asp Val Met Thr Ile Tyr Asn Asp Met 770 775 780 Gln Ala Val Thr Gly Ser Val Ile Ser Met Gly Asn Ile Glu Val Asp 785 790 795 800 Thr Asp Val Glu Leu Gly Met Gly Ser Asn Gly Thr Val Val Phe Ala 805 810 815 Gly Arg Phe Asp Gly Arg Asp Val Ala Val Lys Arg Met Thr Ile Gln 820 825 830 Phe Tyr Asp Ile Ala Thr Arg Glu Thr Lys Leu Leu Arg Glu Ser Asp 835 840 845 Asp His Pro Asn Val Ile Arg Tyr Tyr Ser Gln Val Gln Arg Gly Asp 850 855 860 Phe Leu Tyr Ile Ala Leu Glu Arg Cys Ala Ala Ser Leu Ala Asp Val 865 870 875 880 Ile Glu Lys Pro Tyr Ala Phe Gly Glu Leu Ala Lys Ala Gly Gln Lys 885 890 895 Asp Leu Pro Gly Val Leu Tyr Gln Ile Thr Asn Gly Ile Ser His Leu 900 905 910 His Ser Leu Arg Ile Val His Arg Asp Leu Lys Pro Gln Asn Ile Leu 915 920 925 Val Asn Leu Asp Lys Asp Gly Arg Pro Arg Leu Leu Val Ser Asp Phe 930 935 940 Gly Leu Cys Lys Lys Leu Glu Asp Arg Gln Ser Ser Phe Gly Ala Thr 945 950 955 960 Thr Gly Arg Ala Ala Gly Thr Ser Gly Trp Arg Ala Pro Glu Leu Leu 965 970 975 Leu Asp Asp Asp Gly Gln Asn Pro Ala Ala Ile Asp Ser Ser Thr His 980 985 990 Ser Gly Ser His Thr Ile Leu Val Gly Asp Pro Asn Ser Leu Ser Asn 995 1000 1005 Gly Gly Arg Ala Thr Arg Ala Ile Asp Ile Phe Ser Leu Gly Leu Val 1010 1015 1020 Phe Phe Tyr Val Leu Thr Asn Gly Ser His Pro Phe Asp Cys Gly Asp 1025 1030 1035 1040 Arg Tyr Met Arg Glu Val Asn Ile Arg Lys Gly Asn Tyr Asn Leu Asp 1045 1050 1055 Pro Leu Asp Ala Leu Gly Asp Phe Ala Tyr Glu Ala Lys Asp Leu Ile 1060 1065 1070 Ala Ser Met Leu Gln Ala Ser Pro Lys Ala Arg Pro Asp Ser Arg Glu 1075 1080 1085 Val Met Ala His Pro Phe Phe Trp Ser Pro Lys Lys Arg Leu Ala Phe 1090 1095 1100 Leu Cys Asp Val Ser Asp Ser Leu Glu Lys Glu Val Arg Asp Pro Pro 1105 1110 1115 1120 Pro Ala Leu Val Glu Leu Glu Arg His Ala Pro Glu Val Ile Lys Gly 1125 1130 1135 Asp Phe Leu Lys Val Leu Thr Arg Asp Phe Val Glu Ser Leu Gly Lys 1140 1145 1150 Gln Arg Lys Tyr Thr Gly Asn Lys Leu Leu Asp Leu Leu Arg Ala Leu 1155 1160 1165 Arg Asn Lys Arg Asn His Tyr Glu Asp Met Ser Asp Ser Leu Lys Arg 1170 1175 1180 Ser Val Gly Ser Leu Pro Asp Gly Tyr Leu Ala Tyr Trp Thr Val Lys 1185 1190 1195 1200 Phe Pro Met Leu Leu Leu Thr Cys Trp Asn Val Val Tyr Asn Leu Glu 1205 1210 1215 Trp Glu Lys Thr Asp Arg Phe Arg Glu Tyr Tyr Glu Pro Ala Gly Leu 1220 1225 1230 15 1669 DNA Aspergillus niger 15 ctttttattg ttctatggtt cttaaggaca cctgtccttc ttggccctat ccttcttgtt 60 gtctggtaca cttgacccca ggcaccactt ggccaggcct ggccccccca gcttcccccg 120 ttatgacacg gtggcctgtg ttcctgtgac acgggcaagc agacgtcctc cacaagctgt 180 gtcgacctac atcaccgtcc tcccttgcag tgcggttaag ataaggctca tagtaaatcg 240 attgatccac aattaaagat caatcacctg tcacgcttga aatgatggaa gaagcattct 300 ctccagtcga ctccctcgcc ggctccccga cgcctgagtt gccattgttg acagtgtccc 360 cggcggacac gtcgcttgat gactcgtcag tacaggcagg ggagaccaag gcggaagaga 420 agaagcctgt gaagaagaga aagtcatggg gccaggaatt gccagtcccg aagactaact 480 tgcccccaag gaaacgggcc aagactgaag atgagaaaga gcaacgtcgt atcgagcgcg 540 ttcttcgcaa tcgtgcggca gcacaaacat cacgcgagcg caagaggctc gaaatggaga 600 agttggaaaa tgagaagatt cagatggaac agcaaaacca gttccttctg caacgactat 660 cccagatgga agctgagaac aatcgcttaa accaacaagt cgctcaacta tctgctgagg 720 tccggggctc ccgtggcaac actcccaagc ccggctcccc cgtctcagct tctcctaccc 780 taactcctac cctatttaaa caagaacgcg acgaaatccc tcttgaacgg attcctttcc 840 ccacaccctc tatcaccgac tactccccta ccttgaggcc ttccactctg gctgagtcct 900 ccgacgtgac acaacatcct gcagcggtgt tgtgcgacct gcagtgtccg tcgctggact 960 cgaaggagaa ggaagtgccc tctctctctt tgacgtcggc tcaaaccctg aacctcacgc 1020 tgccgatgat cttgcagctc ctctttctga cgatgacttc caccgcctat tcaacgttga 1080 ttcacccgtt gggtcagatt cttcagtcct tgaagacggg ttcgcctttg acgttctcga 1140 cggaggagat ctatcagcat ttccatttga ttctatggtt gatttcgacc ccgaatctgt 1200 tggcttcgaa ggcatcgagc cgccccacgg tcttccggat gagacttctc gccagacttc 1260 tagcgtgcaa cccagccttg gcgcgtccac ttcgcgatgc gacgggcagg gcattgcagc 1320 tggctgttag cgagcagttt cgccagggag atgcatcggc tgtcgatggt aacggagtcc 1380 aatggagctg ggagtctttg ttgaccttgg cgtggacgat agacctactc gaacagccgg 1440 gacgacgcaa acgaatcttg agcggtttga aatcagcgaa aactggacgg cgaagtaata 1500 ttggcaagtc tcaaaggagt acacggagtt catggagttc acgaagcacc caagaggcgt 1560 tgacgtctct ccttatgggc aagcatagtt gaggttccgg ctgtaaatta tcataaatcc 1620 ttataatttt attctagatt tcaatacagc agttgattgt ctgctcatc 1669 16 386 PRT Aspergillus niger 16 Met Val Leu Lys Asp Thr Cys Pro Ser Trp Pro Tyr Pro Ser Cys Cys 1 5 10 15 Leu Val His Leu Thr Pro Gly Thr Thr Trp Pro Gly Leu Ala Pro Pro 20 25 30 Ala Ser Pro Val Met Thr Arg Trp Pro Val Phe Leu Met Met Glu Glu 35 40 45 Ala Phe Ser Pro Val Asp Ser Leu Ala Gly Ser Pro Thr Pro Glu Leu 50 55 60 Pro Leu Leu Thr Val Ser Pro Ala Asp Thr Ser Leu Asp Asp Ser Ser 65 70 75 80 Val Gln Ala Gly Glu Thr Lys Ala Glu Glu Lys Lys Pro Val Lys Lys 85 90 95 Arg Lys Ser Trp Gly Gln Glu Leu Pro Val Pro Lys Thr Asn Leu Pro 100 105 110 Pro Arg Lys Arg Ala Lys Thr Glu Asp Glu Lys Glu Gln Arg Arg Ile 115 120 125 Glu Arg Val Leu Arg Asn Arg Ala Ala Ala Gln Thr Ser Arg Glu Arg 130 135 140 Lys Arg Leu Glu Met Glu Lys Leu Glu Asn Glu Lys Ile Gln Met Glu 145 150 155 160 Gln Gln Asn Gln Phe Leu Leu Gln Arg Leu Ser Gln Met Glu Ala Glu 165 170 175 Asn Asn Arg Leu Asn Gln Gln Val Ala Gln Leu Ser Ala Glu Val Arg 180 185 190 Gly Ser Arg Gly Asn Thr Pro Lys Pro Gly Ser Pro Val Ser Ala Ser 195 200 205 Pro Thr Leu Thr Pro Thr Leu Phe Lys Gln Glu Arg Asp Glu Ile Pro 210 215 220 Leu Glu Arg Ile Pro Phe Pro Thr Pro Ser Ile Thr Asp Tyr Ser Pro 225 230 235 240 Thr Leu Arg Pro Ser Thr Leu Ala Glu Ser Ser Asp Val Thr Gln His 245 250 255 Pro Ala Val Ser Val Ala Gly Leu Glu Gly Glu Gly Ser Ala Leu Ser 260 265 270 Leu Phe Asp Val Gly Ser Asn Pro Glu Pro His Ala Ala Asp Asp Leu 275 280 285 Ala Ala Pro Leu Ser Asp Asp Asp Phe His Arg Leu Phe Asn Val Asp 290 295 300 Ser Pro Val Gly Ser Asp Ser Ser Val Leu Glu Asp Gly Phe Ala Phe 305 310 315 320 Asp Val Leu Asp Gly Gly Asp Leu Ser Ala Phe Pro Phe Asp Ser Met 325 330 335 Val Asp Phe Asp Pro Glu Ser Val Gly Phe Glu Gly Ile Glu Pro Pro 340 345 350 His Gly Leu Pro Asp Glu Thr Ser Arg Gln Thr Ser Ser Val Gln Pro 355 360 365 Ser Leu Gly Ala Ser Thr Ser Arg Cys Asp Gly Gln Gly Ile Ala Ala 370 375 380 Gly Cys 385 17 20 DNA Aspergillus niger 17 cggtgttgtg cgacctgcag 20 18 44 PRT Aspergillus niger 18 Met Val Leu Lys Asp Thr Cys Pro Ser Trp Pro Tyr Pro Ser Cys Cys 1 5 10 15 Leu Val His Leu Thr Pro Gly Thr Thr Trp Pro Gly Leu Ala Pro Pro 20 25 30 Ala Ser Pro Val Met Thr Arg Trp Pro Val Phe Leu 35 40 19 342 PRT Aspergillus niger 19 Met Met Glu Glu Ala Phe Ser Pro Val Asp Ser Leu Ala Gly Ser Pro 1 5 10 15 Thr Pro Glu Leu Pro Leu Leu Thr Val Ser Pro Ala Asp Thr Ser Leu 20 25 30 Asp Asp Ser Ser Val Gln Ala Gly Glu Thr Lys Ala Glu Glu Lys Lys 35 40 45 Pro Val Lys Lys Arg Lys Ser Trp Gly Gln Glu Leu Pro Val Pro Lys 50 55 60 Thr Asn Leu Pro Pro Arg Lys Arg Ala Lys Thr Glu Asp Glu Lys Glu 65 70 75 80 Gln Arg Arg Ile Glu Arg Val Leu Arg Asn Arg Ala Ala Ala Gln Thr 85 90 95 Ser Arg Glu Arg Lys Arg Leu Glu Met Glu Lys Leu Glu Asn Glu Lys 100 105 110 Ile Gln Met Glu Gln Gln Asn Gln Phe Leu Leu Gln Arg Leu Ser Gln 115 120 125 Met Glu Ala Glu Asn Asn Arg Leu Asn Gln Gln Val Ala Gln Leu Ser 130 135 140 Ala Glu Val Arg Gly Ser Arg Gly Asn Thr Pro Lys Pro Gly Ser Pro 145 150 155 160 Val Ser Ala Ser Pro Thr Leu Thr Pro Thr Leu Phe Lys Gln Glu Arg 165 170 175 Asp Glu Ile Pro Leu Glu Arg Ile Pro Phe Pro Thr Pro Ser Ile Thr 180 185 190 Asp Tyr Ser Pro Thr Leu Arg Pro Ser Thr Leu Ala Glu Ser Ser Asp 195 200 205 Val Thr Gln His Pro Ala Val Ser Val Ala Gly Leu Glu Gly Glu Gly 210 215 220 Ser Ala Leu Ser Leu Phe Asp Val Gly Ser Asn Pro Glu Pro His Ala 225 230 235 240 Ala Asp Asp Leu Ala Ala Pro Leu Ser Asp Asp Asp Phe His Arg Leu 245 250 255 Phe Asn Val Asp Ser Pro Val Gly Ser Asp Ser Ser Val Leu Glu Asp 260 265 270 Gly Phe Ala Phe Asp Val Leu Asp Gly Gly Asp Leu Ser Ala Phe Pro 275 280 285 Phe Asp Ser Met Val Asp Phe Asp Pro Glu Ser Val Gly Phe Glu Gly 290 295 300 Ile Glu Pro Pro His Gly Leu Pro Asp Glu Thr Ser Arg Gln Thr Ser 305 310 315 320 Ser Val Gln Pro Ser Leu Gly Ala Ser Thr Ser Arg Cys Asp Gly Gln 325 330 335 Gly Ile Ala Ala Gly Cys 340 20 36 DNA Artificial Sequence primer 20 atcgcaggat tcccacctac gacaacaacc gccact 36 21 36 DNA Artificial Sequence primer 21 tacagcggat ccctatggat tacgccaatt gtcaag 36 22 72 DNA Artificial Sequence primer 22 ccacctacga caacaaccgc cactatggaa atgactgatt ttgaactact tgcctcgtcc 60 ccgccgggtc ac 72 23 75 DNA Artificial Sequence primer 23 aattataccc tcttgcgatt gtcttcatga agtgatgaag aaatcattga cactggatgg 60 cggcgttagt atcga 75 24 21 DNA Artificial Sequence primer 24 gccatccttg gtgactgagc c 21 25 24 DNA Artificial Sequence primer 25 caattgctcg ctcttacatt gaat 24 26 20 DNA Artificial Sequence primer 26 aattaaccct cactaaaggg 20 27 40 DNA Artificial Sequence primer 27 tggttgatga cgacgatgcg aacagtcatg acaggcaacg 40 28 24 DNA Artificial Sequence HAC1-specific oligonucleotide 28 gggagacgac tgctggaacg ccat 24 29 20 DNA Artificial Sequence primer 29 ccccgagcag tccttgatgg 20 30 17 DNA Artificial Sequence primer 30 gtcgttgatg tcgaagt 17 31 22 DNA Artificial Sequence primer 31 gtaatacgac tcactatagg gc 22 32 21 DNA Artificial Sequence primer 32 ttaggacaga ggccacggtg t 21 33 21 DNA Artificial Sequence primer 33 cccatccttg gtgactgagc c 21 34 21 DNA Artificial Sequence primer 34 aagagtcggt gtcagagttg g 21 35 72 DNA Artificial Sequence primer 35 attaatattt tagcactttg aaaaatgcgt ctacttcgaa gaaacatgct tgcctcgtcc 60 ccgccgggtc ac 72 36 75 DNA Artificial Sequence primer 36 aagcagaggg gcatgaacat gttatgaata caaaaattca cgtaaaatgt cgacactgga 60 tggcggcgtt agtat 75 37 24 DNA Artificial Sequence primer 37 ccgcaacacg acacggcagg caac 24 38 21 DNA Artificial Sequence primer 38 ctaggtagac gttgtatttt g 21 39 36 DNA Artificial Sequence primer 39 tcgaacggat ccgaaaagaa gcccgtcaag aagagg 36 40 39 DNA Artificial Sequence primer 40 atcgcaggat ccctaggttt ggccatcccg cgagccaaa 39 41 38 DNA Artificial Sequence synthetic oligonucleotide 41 cggctgaacc agcgcggcag ccagatgtgg ccaaaggg 38 42 32 DNA Artificial Sequence synthetic oligonucleotide 42 ggtacctgct aaccagcgcg gcatgattca ac 32 43 35 DNA Artificial Sequence synthetic oligonucleotide 43 ggatcttgca tagccagatg tggcctcgat tgact 35 44 33 DNA Artificial Sequence synthetic oligonucleotide 44 ggattagaaa acgccaacgt gtccataacg gtc 33 45 36 DNA Artificial Sequence synthetic oligonucleotide 45 gggcgtggag aagcgagaag tggcctcttc ttctcc 36 46 11 DNA Artificial Sequence binding consensus sequence 46 gcsarngtgk c 11 47 21 DNA Artificial Sequence primer 47 gtggtaatat tacctttaca g 21 48 20 DNA Artificial Sequence primer 48 caatttcaat acgggtggac 20 49 20 DNA Artificial Sequence primer 49 tgtcatcact gctccatctt 20 50 20 DNA Artificial Sequence primer 50 ttaagccttg gcaacatatt 20 51 21 DNA Artificial Sequence primer 51 ttgaacagca gatcgttact g 21 52 21 DNA Artificial Sequence primer 52 tataaagttc gtcaatagtg g 21 53 21 DNA Artificial Sequence primer 53 cggaggcaag agtcatagac g 21 54 23 DNA Artificial Sequence primer 54 caatatattt ctgaaccagt acg 23 55 45 RNA Trichoderma reesei 55 acugauucga cacaacgucc ugcagagaug uugugcgacc cgcag 45 56 45 RNA Aspergillus nidulans 56 cccgauuuga cacaacaucc ugcagcgaug uugugcgacc ugcag 45 57 28 RNA Saccharomyces cerevisiae 57 ccuuguacug uccgaagcgc agucaggu 28 58 60 DNA Trichoderma reesei 58 ccactgattc gacacaacgt cctgcagaga tgttgtgcga cccgcagtgt caatcggtgg 60 59 60 DNA Aspergillus nidulans 59 cccccgattt gacacaacat cctgcagcga tgttgtgcga cctgcagtgt cagtcggcgg 60 60 68 PRT Saccharomyces cerevisiae 60 Lys Ser Thr Leu Pro Pro Arg Lys Arg Ala Lys Thr Lys Glu Glu Lys 1 5 10 15 Glu Gln Arg Arg Ile Glu Arg Ile Leu Arg Asn Arg Arg Ala Ala His 20 25 30 Gln Ser Arg Glu Lys Lys Arg Leu His Leu Gln Tyr Leu Glu Arg Lys 35 40 45 Cys Ser Leu Leu Glu Asn Leu Leu Asn Ser Val Asn Leu Glu Lys Leu 50 55 60 Ala Asp His Glu 65 61 12 DNA Trichoderma reesei 61 gccagatgtg gc 12 62 11 DNA Trichoderma reesei 62 gccaacgtgt c 11 63 12 DNA Trichoderma reesei 63 gcgagaagtg gc 12 

What is claimed is:
 1. A method of increasing the secretion of a heterologous protein in a eukaryotic cell comprising inducing an elevated unfolded protein response (UPR).
 2. The method of claim 1 wherein inducing is by increasing the presence of HAC1 protein in said cell.
 3. The method of claim 2 wherein said HAC1 protein is constitutively produced.
 4. The method of claim 2 wherein said increase of HAC1 protein is by a UPR inducing form of a HAC1 recombinant nucleic acid.
 5. The method of claim 2 wherein said HAC1 protein is encoded by a nucleic acid isolated from a cell selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Fusarium, Neurospora, and Penicillium.
 6. The method of claim 2 wherein said HAC1 protein is encoded by a nucleic acid isolated from yeast.
 7. The method of claim 6 wherein said yeast is Saccharomyces cerevisiae.
 8. The method of claim 2 wherein said HAC1 protein is encoded by a nucleic acid isolated from filamentous fungi.
 9. The method of claim 8 wherein said fungi is from Trichoderma.
 10. The method of claim 8 wherein said fungi is Trichoderma reesei.
 11. The method of claim 8 wherein said fungi is from Aspergillus.
 12. The method of claim 8 wherein said fungi is Aspergillus nidulans.
 13. The method of claim 8 wherein said fungi is Aspergillus niger.
 14. The method of claim 1 wherein said inducing is by modulating the level of IRE1 protein or PTC2 protein in said cell.
 15. The method of claim 1 wherein said inducing is by increasing the level of IRE1 protein.
 16. The method of claim 15 wherein said IRE1 is an IRE1 variant which has the activity of a constitutively phosphorylated IRE1.
 17. The method of claim 15 wherein said IRE1 protein is encoded by a nucleic acid isolated from a cell selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Fusarium, Neurospora, and Penicillium.
 18. The method of claim 15 wherein said IRE1 protein is encoded by a nucleic acid isolated from yeast.
 19. The method of claim 18 wherein said yeast is Saccharomyces cerevisiae.
 20. The method of claim 15 wherein said IRE1 is isolated from filamentous fungi.
 21. The method of claim 20 wherein said fungi is from Trichoderma.
 22. The method of claim 20 wherein said fungi is Trichoderma reesei.
 23. The method of claim 20 wherein said fungi is from Aspergillus.
 24. The method of claim 20 wherein said fungi is Aspergillus nidulans.
 25. The method of claim 20 wherein said fungi is Aspergillus niger.
 26. The method of claim 1 wherein said cell is selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Fusarium, Neurospora, and Penicillium.
 27. The method of claim 1 wherein said cell is a yeast cell.
 28. The method of claim 27 wherein said yeast is Saccharomyces cerevisiae.
 29. The method of claim 1 wherein said cell is from filamentous fungi.
 30. The method of claim 29 wherein said fungi is from Trichoderma.
 31. The method of claim 29 wherein said fungi is Trichoderma reesei.
 32. The method of claim 29 wherein said fungi is from Aspergillus.
 33. The method of claim 29 wherein said fungi is Aspergillus nidulans.
 34. The method of claim 29 wherein said fungi is Aspergillus niger.
 35. The method of claim 1 wherein said cell is an insect cell.
 36. The method of claim 1 wherein said cell is a mammalian cell.
 37. An isolated nucleic acid encoding a HAC1 protein, wherein said HAC1 protein induces unfolded protein response and has less than 50% similarity to yeast HAC1 protein.
 38. An isolated nucleic acid encoding a HAC1 protein, wherein said HAC1 protein induces unfolded protein response and wherein said HAC1 protein comprises a DNA binding region that has greater than 70% similarity to the DNA binding region of filamentous fungi HAC1 protein.
 39. The nucleic acid of claim 38 wherein said filamentous fungi HAC1 protein has an amino acid sequence as shown in FIG. 7 or FIG. 8 or FIG.
 28. 40. The nucleic acid of claim 38 wherein said HAC1 protein has an amino acid sequence having greater than 70% similarity to the sequence of FIG. 7 or FIG. 8 or FIG.
 28. 41. The nucleic acid of claim 38 isolated from Trichoderma reesel.
 42. The nucleic acid of claim 38 isolated from Aspergillus nidulans.
 43. The nucleic acid of claim 38 isolated from Aspergillus niger.
 44. The nucleic acid of claim 38 wherein said HAC1 protein has an amino acid sequence as set forth in FIG.
 7. 45. The nucleic acid of claim 38 wherein said HAC1 protein has an amino acid sequence as set forth in FIG.
 8. 46. The nucleic acid of claim 38 wherein said HAC1 protein has an amino acid sequence as set forth in FIG.
 28. 47. The nucleic acid of claim 38 wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG.
 7. 48. The nucleic acid of claim 38 wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG.
 7. 49. The nucleic acid of claim 38 wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG.
 8. 50. The nucleic acid of claim 38 wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG.
 8. 51. The nucleic acid of claim 38 wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG.
 28. 52. The nucleic acid of claim 38 wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG.
 28. 53. A protein encoded by the nucleic acid of claim
 37. 54. A protein having unfolded protein response inducing activity and having greater than 70% similarity to an amino acid sequence of FIG. 7 or FIG. 8 or FIG.
 28. 55. A protein having an amino acid sequence as set forth in FIG. 7 or FIG. 8 or FIG.
 28. 56. An isolated nucleic acid encoding a PTC2 protein wherein said PTC2 protein modulates unfolded protein response and wherein said PTC2 has at least 70% similarity to an amino acid sequence of FIG. 24 or FIG.
 25. 57. The nucleic acid of claim 56 isolated from Trichoderma reesei.
 58. The nucleic acid of claim 56 isolated from Aspergillus nidulans.
 59. The nucleic acid of claim 56 isolated from Aspergillus niger.
 60. The nucleic acid of claim 56 wherein said PTC2 protein has an amino acid sequence as set forth in FIG.
 24. 61. The nucleic acid of claim 56 wherein said PTC2 protein has an amino acid sequence as set forth in FIG.
 25. 62. The nucleic acid of claim 56 wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG.
 24. 63. The nucleic acid of claim 56 wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG.
 24. 64. The nucleic acid of claim 56 wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG.
 25. 65. A protein encoded by the nucleic acid of claim
 56. 66. A protein having unfolded protein response modulating activity and having greater than 70% similarity to the amino acid sequence of FIG. 24 or FIG.
 25. 67. A protein having an amino acid sequence as set forth in FIG. 24 or FIG.
 25. 68. A nucleic acid encoding a IRE1 protein having unfolded protein response modulating activity and having at least 60% to an amino acid sequence as shown in FIG. 26 or FIG.
 27. 69. The nucleic acid of claim 68 wherein said IRE1 protein has an amino acid sequence as shown in FIG. 26 or FIG.
 27. 70. The nucleic acid of claim 68 wherein said nucleic acid is isolated from Trichoderma reesei.
 71. The nucleic acid of claim 68 wherein said nucleic acid is isolated from Aspergillus nidulans.
 72. The nucleic acid of claim 68 wherein said nucleic acid is isolated from Aspergillus niger.
 73. The nucleic acid of claim 68 wherein said IRE1 protein has an amino acid sequence as set forth in FIG.
 26. 74. The nucleic acid of claim 68 wherein said IRE1 protein has an amino acid sequence as set forth in FIG.
 27. 75. The nucleic acid of claim 68 wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG.
 26. 76. The nucleic acid of claim 68 wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG.
 26. 77. The nucleic acid of claim 68 wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG.
 27. 78. The nucleic acid of claim 68 wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG.
 27. 79. A protein encoded by the nucleic acid of claim
 68. 80. A protein having unfolded protein response inducing activity and having greater than 70% similarity to the amino acid sequence of FIG. 26 or FIG.
 27. 81. The protein of claim 80 wherein said protein has constitutive unfolded protein response inducing activity.
 82. A protein having an amino acid sequence as set forth in FIG. 26 or FIG.
 27. 83. A cell containing a heterologous nucleic acid encoding a protein having unfolded protein response modulating activity and a heterologous nucleic acid encoding a protein of interest to be secreted.
 84. The cell of claim 83 wherein said protein having unfolded protein response modulating activity is selected from the proteins selected from the group consisting of HAC1, PTC2 and IRE1.
 85. The cell of claim 83 wherein said protein of interest is selected from the group consisting of lipase, cellulase, endo-glucosidase H, protease, carbohydrase, reductase, oxidase, isomerase, transferase, kinase, phosphatase, alpha-amylase, glucoamylase, lignocellulose hemicellulase, pectinase and ligninase.
 86. A protein encoded by the nucleic acid of claim
 38. 